Fermentative glycerol-free ethanol production

ABSTRACT

The present invention relates to a yeast cell, in particular a recombinant yeast cell, the cell lacking enzymatic activity needed for the NADH-dependent glycerol synthesis or the cell having a reduced enzymatic activity with respect to the NADH-dependent glycerol synthesis compared to its corresponding wild-type yeast cell, the cell comprising one or more heterologous nucleic acid sequences encoding an NAD+-dependent acetylating acetaldehyde dehydrogenase (EC 1.2.1.10) activity. The invention further relates to the use of a cell according to the invention in the preparation of ethanol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/970,641, filed 3May 2018, which is a continuation of U.S. Ser. No. 15/154,199, filed 13May 2016, which is a continuation of U.S. Ser. No. 14/189,989, filed 25Feb. 2014, now U.S. Pat. No. 9,528,117, which is a continuation of U.S.Ser. No. 13/061,695, filed 18 Jul. 2011, now U.S. Pat. No. 8,795,998,which is a national phase of PCT/NL2010/050475, filed 23 Jul. 2010,which claims benefit of European patent application No. 09166360.9,filed 24 Jul. 2009. The contents of the above patent applications areincorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name:2919208-306005_Sequence_Listing_ST25.txt date created: 27 Mar. 2020,size: 38,859 bytes).

BACKGROUND OF THE DISCLOSURE

The present invention relates to a recombinant yeast cell having theability to produce a desired fermentation product, to the constructionof said yeast cell by genetic modification and to a process forproducing a fermentation product wherein said yeast cell is used.

Ethanol production by Saccharomyces cerevisiae is currently, by volume,the single largest fermentation process in industrial biotechnology. Aglobal research effort is underway to expand the substrate range of S.cerevisiae to include lignocellulosic hydrolysates, in particularhydrolysed lignocellulosic biomass from non-food feedstocks (e.g. energycrops and agricultural residues, forestry residues orindustrial/consumer waste materials that are rich in cellulose,hemicellulose and/or pectin) and to increase productivity, robustnessand product yield.

Lignocellulosic biomass is abundant, however is in general not readilyfermented by wild-type ethanol producing micro-organisms, such as S.cerevisiae. The biomass has to be hydrolysed. The resultant hydrolysateis often a mixture of various monosaccharides and oligosaccharides,which may not all be suitable substrates for the wild-typemicro-organism. Further, the hydrolysates typically comprise aceticacid, formed as a by-product in particular when hydrolysing pectin orhemicellulose, and—dependent on the type of hydrolysis—one or more otherby-products or residual reagents that may adversely affect thefermentation. In particular, acetic acid has been reported to negativelyaffect the kinetics and/or stoichiometry of sugar fermentation bywild-type and genetically modified S. cerevisiae strains and itstoxicity is strongly augmented at low culture pH (Helle et al. EnzymeMicrob Technol 33 (2003) 786-792; Bellissimi et al. FEMS Yeast Res 9(2009) 358-364).

Various approaches have been proposed to improve the fermentativeproperties of ethanol producing organisms by genetic modification, andto improve the hydrolysis process of the biomass. E.g. an overview ofdevelopments in the fermentative production of ethanol from biomasshydrolysates is given in a review by A. van Maris et al. (Antonie vanLeeuwenhoek (2006) 90:391-418). Reference is made to various ways inwhich S. cerevisiae may be modified and to various methods ofhydrolysing lignocellulosic biomass.

A major challenge relating to the stoichiometry of yeast based ethanolproduction is that substantial amounts of glycerol are invariably formedas a by-product. It has been estimated that, in typical industrialethanol processes, up to about 4 wt. % of the sugar feedstock isconverted into glycerol (Nissen et al. Yeast 16 (2000) 463-474). Underconditions that are ideal for anaerobic growth, the conversion intoglycerol may even be higher, up to about 10%.

Glycerol production under anaerobic conditions is primarily linked toredox metabolism. During anaerobic growth of S. cerevisiae, sugardissimilation occurs via alcoholic fermentation. In this process, theNADH formed in the glycolytic glyceraldehyde-3-phosphate dehydrogenasereaction is reoxidized by converting acetaldehyde, formed bydecarboxylation of pyruvate to ethanol via NAD⁺-dependent alcoholdehydrogenase. The fixed stoichiometry of this redox-neutraldissimilatory pathway causes problems when a net reduction of NAD⁺ toNADH occurs elsewhere in metabolism. Under anaerobic conditions, NADHreoxidation in S. cerevisiae is strictly dependent on reduction of sugarto glycerol. Glycerol formation is initiated by reduction of theglycolytic intermediate dihydroxyacetone phosphate to glycerol3-phosphate, a reaction catalyzed by NAD⁺-dependent glycerol 3-phosphatedehydrogenase. Subsequently, the glycerol 3-phosphate formed in thisreaction is hydrolysed by glycerol-3-phosphatase to yield glycerol andinorganic phosphate. Consequently, glycerol if a major by-product duringanaerobic production of ethanol by S. cerevisiae, which is undesired asit reduces overall conversion of sugar to ethanol. Further, the presenceof glycerol in effluents of ethanol production plants may impose costsfor waste-water treatment.

It is an object of the invention to provide a novel recombinant cell,which is suitable for the anaerobic, fermentative production of ethanolfrom a carbohydrate, in particular a carbohydrate obtained fromlignocellulosic biomass, which has a reduced glycerol productioncompared to its corresponding wild-type organism or which lacks glycerolproduction if the cell is used for the fermentative preparation ofethanol.

It is further an object to provide a novel method for fermentativelypreparing ethanol in anaerobic yeast cultures, in which method noglycerol is formed, or at least wherein less glycerol is formed than ina method making use of known strains of S. cerevisiae.

One or more further objects that may be met are apparent from thedescription and/or claims.

The inventors have realized that it is possible to meet one or more ofthese objectives by providing a specific recombinant cell wherein aspecific other enzymatic activity has been incorporated, which allowsre-oxidation of NADH formed in the fermentation of a carbohydrate, alsoin the absence of enzymatic activity needed for the NADH-dependentglycerol synthesis.

Accordingly the present invention relates to a recombinant yeast cell,the cell comprising one or more recombinant, in particular one or moreheterologous, nucleic acid sequences encoding an NAD⁺-dependentacetylating acetaldehyde dehydrogenase (EC 1.2.1.10) activity.

The inventors have in particular realized that it is advantageous toprovide a cell without enzymatic activity needed for the NADH-dependentglycerol synthesis or a cell with reduced enzymatic activity needed forthe NADH-dependent glycerol synthesis.

Accordingly, the invention in particular relates to a recombinant yeastcell comprising one or more heterologous nucleic acid sequences encodingan NAD⁺-dependent acetylating acetaldehyde dehydrogenase activity,wherein the cell lacks enzymatic activity needed for the NADH-dependentglycerol synthesis (i.e. is free of such activity), or wherein the cellhas a reduced enzymatic activity with respect to NADH-dependent glycerolsynthesis compared to its corresponding wild-type yeast cell.

The invention is further directed to the use of a cell according to theinvention for the preparation of ethanol.

In particular, the invention is further directed to a method forpreparing ethanol, comprising preparing ethanol from a fermentablecarbohydrate and from acetate, which preparation is carried out underanaerobic fermentative conditions using a yeast cell, said cellexpressing acetyl-Coenzyme A synthetase activity and NAD⁺-dependentacetylating acetaldehyde dehydrogenase activity, said cell preferablylacking enzymatic activity needed for the biochemical pathway forglycerol synthesis from a carbohydrate or having a reduced enzymaticactivity with respect to the biochemical pathway for glycerol synthesisfrom a carbohydrate compared to a wild-type S. cerevisiae cell.

Advantageously, in accordance with the invention ethanol is produced ina molar ratio of glycerol:ethanol of less than 0.04:1, in particular ofless than 0.02:1, preferably of less than 0.01:1. Glycerol productionmay be absent (undetectable), although at least in some embodiments(wherein NADH-dependent glycerol synthesis is reduced yet not completelyprohibited) some glycerol may be produced as a side-product, e.g. in aratio glycerol to ethanol of 0.001:1 or more.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows a genetic modification procedure which may becarried out as part of the production of a cell according to theinvention.

FIG. 2 shows concentrations of biomass and products in anaerobic batchcultures of different S. cerevisiae strains on glucose (20 g l⁻¹).Acetic acid (2.0 g l⁻¹) was present from the start of the fermentation(panel A, B) or added at the time point indicated by the arrow (panel C,D). Growth conditions: T=30° C., pH 5.0. Symbols: ▴, opticcc

csity ct 660 cm; ●, glucose;

◯, ethanol; ▪, acetate; □, glycerol. Each graph represents values fromone of two independent replicates, yielding data that differed by lessthan 5%. Panel A: S. cerevisiae IME076 (GPD1 GPD2). Panel B: S.cerevisiae IMZ132 (gpd1Δ gpd2Δ overexpressing the E. coli mhpF gene).Panel C: S. cerevisiae IMZ132 (gpd1Δ gpd2Δ overexpressing the E. colimhpF gene). Panel D: S. cerevisiae IMZ127 (gpd1Δ gpd2Δ) grown on glucose(20 g·l⁻¹).

DETAILED DESCRIPTION OF THE DISCLOSURE

The present invention allows complete elimination of glycerolproduction, or at least a significant reduction thereof, by providing arecombinant yeast cell, in particular S. cerevisiae, such that it canreoxidize NADH by the reduction of acetic acid to ethanol viaNADH-dependent reactions.

This is not only advantageous in that glycerol production is avoided orat least reduced, but since the product formed in the re-oxidation ofNADH is also the desired product, namely ethanol, a method of theinvention may also offer an increased product yield (determined as thewt. % of converted feedstock, i.e. carbohydrate plus acetic acid, thatis converted into ethanol).

Since acetic acid is generally available at significant amounts inlignocellulosic hydrolysates, this makes the present inventionparticularly advantageous for the preparation of ethanol usinglignocellulosic biomass as a source for the fermentable carbohydrate.Further, carbohydrate sources that may contain a considerable amount ofacetate include sugar beet molasses (hydrolysates of) and starchcontaining (e.g. waste products from corn dry milling processes, fromcorn wet milling processes; from starch wastes processes, e.g. withstillage recycles). The invention contributes to a decrease of thelevels of the inhibiting compound acetic acid and a larger fraction ofthe hydrolysate actually becomes a substrate for the production of theethanol.

Good results have been achieved with a yeast cell without noticeableenzymatic activity needed for the NADH-dependent glycerol synthesis, asillustrated in the example. However, the inventors contemplate that alsoa yeast cell according to the invention having NADH-dependent glycerolsynthesis activity may advantageously be used for, e.g., ethanolproduction. It is contemplated that such cell can use acetate tore-oxidize at least part of the NADH. Thereby the acetate may competewith the NADH-dependent glycerol synthesis pathway and thus potentiallyreduce the glycerol synthesis. Moreover, acetate present in a feedstockused for the production of ethanol, such as a lignocellulosichydrolysate, can be converted into ethanol, thereby increasing productyield.

The term “a” or “an” as used herein is defined as “at least one” unlessspecified otherwise.

When referring to a noun (e.g. a compound, an additive, etc.) in thesingular, the plural is meant to be included. Thus, when referring to aspecific moiety, e.g. “compound”, this means “at least one” of thatmoiety, e.g. “at least one compound”, unless specified otherwise.

The term ‘or’ as used herein is to be understood as ‘and/or’.

When referred herein to a carboxylate, e.g. acetate, the correspondingcarboxylic acid (its conjugated acid) as well as a salt thereof is meantto be included, and vice versa.

When referring to a compound of which several isomers exist (e.g. a Dand an L enantiomer), the compound in principle includes allenantiomers, diastereomers and cis/trans isomers of that compound thatmay be used in the particular method of the invention; in particularwhen referring to such as compound, it includes the natural isomer(s).

The term ‘fermentation’, ‘fermentative’ and the like is used herein in aclassical sense, i.e. to indicate that a process is or has been carriedout under anaerobic conditions. Anaerobic conditions are herein definedas conditions without any oxygen or in which essentially no oxygen isconsumed by the yeast cell, in particular a yeast cell, and usuallycorresponds to an oxygen consumption of less than 5 mmol/l·h, inparticular to an oxygen consumption of less than 2.5 mmol/l·h, or lessthan 1 mmol/l·h. This usually corresponds to a dissolved oxygenconcentration in the culture broth of less than 5% of air saturation, inparticular to a dissolved oxygen concentration of less than 1% of airsaturation, or less than 0.2% of air saturation.

The term “yeast” or “yeast cell” refers to a phylogenetically diversegroup of single-celled fungi, most of which are in the division ofAscomycota and Basidiomycota. The budding yeasts (“true yeasts”) areclassified in the order Saccharomycetales, with Saccharomyces cerevisiaeas the most well-known species.

The term “recombinant (cell)” as used herein, refers to a strain (cell)containing nucleic acid which is the result of one or more geneticmodifications using recombinant DNA technique(s) and/or anothermutagenic technique(s). In particular a recombinant cell may comprisenucleic acid not present in a corresponding wild-type cell, whichnucleic acid has been introduced into that strain (cell) usingrecombinant DNA techniques (a transgenic cell), or which nucleic acidnot present in said wild-type is the result of one or more mutations forexample using recombinant DNA techniques or another mutagenesistechnique such as UV-irradiation in a nucleic acid sequence present insaid wild-type (such as a gene encoding a wild-type polypeptide) orwherein the nucleic acid sequence of a gene has been modified to targetthe polypeptide product (encoding it) towards another cellularcompartment. Further, the term “recombinant (cell)” in particularrelates to a strain (cell) from which DNA sequences have been removedusing recombinant DNA techniques.

The term “transgenic (yeast) cell” as used herein, refers to a strain(cell) containing nucleic acid not naturally occurring in that strain(cell) and which has been introduced into that strain (cell) usingrecombinant DNA techniques, i.e. a recombinant cell).

The term “mutated” as used herein regarding proteins or polypeptidesmeans that at least one amino acid in the wild-type or naturallyoccurring protein or polypeptide sequence has been replaced with adifferent amino acid, inserted or deleted from the sequence viamutagenesis of nucleic acids encoding these amino acids. Mutagenesis isa well-known method in the art, and includes, for example, site-directedmutagenesis by means of PCR or is oligonucleotide-mediated mutagenesisas described in Sambrook et al., Molecular Cloning—A Laboratory Manual,2nd ed., Vol. 1-3 (1989). The term “mutated” as used herein regardinggenes means that at least one nucleotide in the nucleic acid sequence ofthat gene or a regulatory sequence thereof, has been replaced with adifferent nucleotide, or has been deleted from the sequence viamutagenesis, resulting in the transcription of a protein sequence with aqualitatively of quantitatively altered function or the knock-out ofthat gene.

The term “gene”, as used herein, refers to a nucleic acid sequencecontaining a template for a nucleic acid polymerase, in eukaryotes, RNApolymerase II. Genes are transcribed into mRNAs that are then translatedinto protein.

The term “nucleic acid” as used herein, includes reference to adeoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, ineither single or double-stranded form, and unless otherwise limited,encompasses known analogues having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to naturally occurring nucleotides (e.g., peptide nucleicacids). A polynucleotide can be full-length or a subsequence of a nativeor heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulphation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation.

When an enzyme is mentioned with reference to an enzyme class (EC), theenzyme class is a class wherein the enzyme is classified or may beclassified, on the basis of the Enzyme Nomenclature provided by theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (NC-IUBMB), which nomenclature may be found on theWorld Wide Web at chem.qmul.ac.uk/iubmb/enzyme/. Other suitable enzymesthat have not (yet) been classified in a specified class but may beclassified as such, are meant to be included.

If referred herein to a protein or a nucleic acid sequence, such as agene, by reference to a accession number, this number in particular isused to refer to a protein or nucleic acid sequence (gene) having asequence as can be found on the World Wide Web at ncbi.nlm.nih.gov/, (asavailable on 13 Jul. 2009) unless specified otherwise.

Every nucleic acid sequence herein that encodes a polypeptide also, byreference to the genetic code, describes every possible silent variationof the nucleic acid. The term “conservatively modified variants” appliesto both amino acid and nucleic acid sequences. With respect toparticular nucleic acid sequences, conservatively modified variantsrefers to those nucleic acids which encode identical or conservativelymodified variants of the amino acid sequences due to the degeneracy ofthe genetic code. The term “degeneracy of the genetic code” refers tothe fact that a large number of functionally identical nucleic acidsencode any given protein. For instance, the codons GCA, GCC, GCG and GCUall encode the amino acid alanine. Thus, at every position where analanine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations” and represent onespecies of conservatively modified variation.

The term “functional homologue” (or in short “homologue”) of apolypeptide having a specific sequence (e.g. SEQ ID NO: 2), as usedherein, refers to a polypeptide comprising said specific sequence withthe proviso that one or more amino acids are substituted, deleted,added, and/or inserted, and which polypeptide has (qualitatively) thesame enzymatic functionality for substrate conversion, for instance anNAD⁺-dependent acetylating acetaldehyde dehydrogenase (EC 1.2.1.10)activity homologue is capable of converting acetaldehyde into ethanol.This functionality may be tested by use of an assay system comprising arecombinant yeast cell comprising an expression vector for theexpression of the homologue in yeast, said expression vector comprisinga heterologous nucleic acid sequence operably linked to a promoterfunctional in the yeast and said heterologous nucleic acid sequenceencoding the homologous polypeptide of which enzymatic activity forconverting acetyl-Coenzyme A to acetaldehyde in the yeast cell is to betested, and assessing whether said conversion occurs in said cells.Candidate homologues may be identified by using in silico similarityanalyses. A detailed example of such an analysis is described in Example2 of WO2009/013159. The skilled person will be able to derive there fromhow suitable candidate homologues may be found and, optionally uponcodon (pair) optimization, will be able to test the requiredfunctionality of such candidate homologues using a suitable assay systemas described above. A suitable homologue represents a polypeptide havingan amino acid sequence similar to a specific polypeptide of more than50%, preferably of 60% or more, in particular of at least 70%, more inparticular of at least 80%, at least 90%, at least 95%, at least 97%, atleast 98% or at least 99%, for instance having such an amino acidsequence similarity to SEQ ID NO: 2, and having the required enzymaticfunctionality for converting acetyl-Coenzyme A to acetaldehyde. Withrespect to nucleic acid sequences, the term functional homologue ismeant to include nucleic acid sequences which differ from anothernucleic acid sequence due to the degeneracy of the genetic code andencode the same polypeptide sequence.

Sequence identity is herein defined as a relationship between two ormore amino acid (polypeptide or protein) sequences or two or morenucleic acid (polynucleotide) sequences, as determined by comparing thesequences. Usually, sequence identities or similarities are comparedover the whole length of the sequences compared. In the art, “identity”also means the degree of sequence relatedness between amino acid ornucleic acid sequences, as the case may be, as determined by the matchbetween strings of such sequences.

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include e.g. the BestFit, BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990),publicly available from NCBI and other sources (BLAST Manual, Altschul,S., et al., NCBI NLM NIH Bethesda, Md. 20894). Preferred parameters foramino acid sequences comparison using BLASTP are gap open 11.0, gapextend 1, Blosum 62 matrix.

“Expression” refers to the transcription of a gene into structural RNA(rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into aprotein.

As used herein, “heterologous” in reference to a nucleic acid or proteinis a nucleic acid or protein that originates from a foreign species, or,if from the same species, is substantially modified from its native formin composition and/or genomic locus by deliberate human intervention.For example, a promoter operably linked to a heterologous structuralgene is from a species different from that from which the structuralgene was derived, or, if from the same species, one or both aresubstantially modified from their original form. A heterologous proteinmay originate from a foreign species or, if from the same species, issubstantially modified from its original form by deliberate humanintervention.

The term “heterologous expression” refers to the expression ofheterologous nucleic acids in a host cell. The expression ofheterologous proteins in eukaryotic host cell systems such as yeast arewell known to those of skill in the art. A polynucleotide comprising anucleic acid sequence of a gene encoding an enzyme with a specificactivity can be expressed in such a eukaryotic system. In someembodiments, transformed/transfected yeast cells may be employed asexpression systems for the expression of the enzymes. Expression ofheterologous proteins in yeast is well known. Sherman, F., et al.,Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is awell recognized work describing the various methods available to expressproteins in yeast. Two widely utilized yeasts are Saccharomycescerevisiae and Pichia pastoris. Vectors, strains, and protocols forexpression in Saccharomyces and Pichia are known in the art andavailable from commercial suppliers (e.g., Invitrogen). Suitable vectorsusually have expression control sequences, such as promoters, including3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like as desired.

As used herein “promoter” is a DNA sequence that directs thetranscription of a (structural) gene. Typically, a promoter is locatedin the 5′-region of a gene, proximal to the transcriptional start siteof a (structural) gene. Promoter sequences may be constitutive,inducible or repressible. If a promoter is an inducible promoter, thenthe rate of transcription increases in response to an inducing agent.

The term “vector” as used herein, includes reference to an autosomalexpression vector and to an integration vector used for integration intothe chromosome.

The term “expression vector” refers to a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestunder the control of (i.e. operably linked to) additional nucleic acidsegments that provide for its transcription. Such additional segmentsmay include promoter and terminator sequences, and may optionallyinclude one or more origins of replication, one or more selectablemarkers, an enhancer, a polyadenylation signal, and the like. Expressionvectors are generally derived from plasmid or viral DNA, or may containelements of both. In particular an expression vector comprises a nucleicacid sequence that comprises in the 5′ to 3′ direction and operablylinked: (a) a yeast-recognized transcription and translation initiationregion, (b) a coding sequence for a polypeptide of interest, and (c) ayeast-recognized transcription and translation termination region.“Plasmid” refers to autonomously replicating extrachromosomal DNA whichis not integrated into a microorganism's genome and is usually circularin nature.

An “integration vector” refers to a DNA molecule, linear or circular,that can be incorporated in a microorganism's genome and provides forstable inheritance of a gene encoding a polypeptide of interest. Theintegration vector generally comprises one or more segments comprising agene sequence encoding a polypeptide of interest under the control of(i.e. operably linked to) additional nucleic acid segments that providefor its transcription. Such additional segments may include promoter andterminator sequences, and one or more segments that drive theincorporation of the gene of interest into the genome of the targetcell, usually by the process of homologous recombination. Typically, theintegration vector will be one which can be transferred into the targetcell, but which has a replicon which is nonfunctional in that organism.Integration of the segment comprising the gene of interest may beselected if an appropriate marker is included within that segment.

As used herein, the term “operably linked” refers to a juxtapositionwherein the components so described are in a relationship permittingthem to function in their intended manner. A control sequence “operablylinked” to another control sequence and/or to a coding sequence isligated in such a way that transcription and/or expression of the codingsequence is achieved under conditions compatible with the controlsequence. Generally, operably linked means that the nucleic acidsequences being linked are contiguous and, where necessary to join twoprotein coding regions, contiguous and in the same reading frame.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells. Preferably, host cells are cellsof the order of Actinomycetales, most preferably yeast cells, mostpreferably cells of Saccharomyces cerevisiae.

“Transformation” and “transforming”, as used herein, refers to theinsertion of an exogenous polynucleotide into a host cell, irrespectiveof the method used for the insertion, for example, direct uptake,transduction, f-mating or electroporation. The exogenous polynucleotidemay be maintained as a non-integrated vector, for example, a plasmid, oralternatively, may be integrated into the host cell genome.

A cell according to the invention preferably is selected from the groupof Saccharomycetaceae, more preferably from the group of Saccharomycescells, Zygosaccharomyces and Kluyveromyces cells. In particular goodresults have been achieved with a Saccharomyces cerevisiae host cell.Zygosaccharomyces baillii is another particularly preferred cell,especially for its high acetate tolerance and its high ethanoltolerance.

Further, a cell according to the invention may be a yeast cell selectedfrom the group of xylose-fermenting yeasts, more preferably a Pichiaspecies, for example Pichia stipitis or Pichia angusta (also known asHansenula polymorpha).

In a further embodiment, a host cell according to the invention is ahost cell that naturally lacks enzymatic activity needed for theNADH-dependent glycerol synthesis, for example yeast cells belonging tothe species Brettanomyces intermedius.

A preferred cell according to the invention is free of enzymaticactivity needed for the NADH-dependent glycerol synthesis or has areduced enzymatic activity with respect to the NADH-dependentbiochemical pathway for glycerol synthesis from a carbohydrate comparedto its corresponding wild-type yeast cell.

A reduced enzymatic activity can be achieved by modifying one or moregenes encoding a NAD-dependent glycerol 3-phosphate dehydrogenaseactivity (GPD) or one or more genes encoding a glycerol phosphatephosphatase activity (GPP), such that the enzyme is expressedconsiderably less than in the wild-type or such that the gene encoded apolypeptide with reduced activity. Such modifications can be carried outusing commonly known biotechnological techniques, and may in particularinclude one or more knock-out mutations or site-directed mutagenesis ofpromoter regions or coding regions of the structural genes encoding GPDand/or GPP. Alternatively, yeast strains that are defective in glycerolproduction may be obtained by random mutagenesis followed by selectionof strains with reduced or absent activity of GPD and/or GPP. S.cerevisiae GDP 1, GDP2, GPP1 and GPP2 genes are shown in SEQ ID NO:24-27.

Preferably at least one gene encoding a GPD or at least one geneencoding a GPP is entirely deleted, or at least a part of the gene isdeleted that encodes a part of the enzyme that is essential for itsactivity. In particular, good results have been achieved with a S.cerevisiae cell, wherein the open reading frames of the GPD1 gene and ofthe GPD2 gene have been inactivated. Inactivation of a structural gene(target gene) can be accomplished by a person skilled in the art bysynthetically synthesizing or otherwise constructing a DNA fragmentconsisting of a selectable marker gene flanked by DNA sequences that areidentical to sequences that flank the region of the host cell's genomethat is to be deleted. In particular, good results have been obtainedwith the inactivation of the GPD1 and GPD2 genes in Saccharomycescerevisiae by integration of the marker genes kanMX and hphMX4.Subsequently this DNA fragment is transformed into a host cell.Transformed cells that express the dominant marker gene are checked forcorrect replacement of the region that was designed to be deleted, forexample by a diagnostic polymerase chain reaction or Southernhybridization.

As indicated above, a cell according to the invention comprises aheterologous nucleic acid sequence encoding an NAD⁺-dependent,acetylating acetaldehyde dehydrogenase (EC 1.2.1.10). This enzymecatalyses the conversion of acetyl-Coenzyme A to acetaldehyde. Thisconversion can be represented by the equilibrium reaction formula:acetyl-Coenzyme A+NADH+H⁺↔acetaldehyde+NAD⁺+Coenzyme A.

Thus, this enzyme allows the re-oxidation of NADH when acetyl-Coenzyme Ais generated from acetate present in the growth medium, and therebyglycerol synthesis is no longer needed for redox cofactor balancing.

The nucleic acid sequence encoding the NAD⁺-dependent acetylatingacetaldehyde dehydrogenase may in principle originate from any organismcomprising a nucleic acid sequence encoding said dehydrogenase.

Known NAD⁺-dependent acetylating acetaldehyde dehydrogenases that cancatalyse the NADH-dependent reduction of acetyl-Coenzyme A toacetaldehyde may in general be divided in three types of NAD⁺-dependentacetylating acetaldehyde dehydrogenase functional homologues:

1) Bifunctional proteins that catalyse the reversible conversion ofacetyl-Coenzyme A to acetaldehyde, and the subsequent reversibleconversion of acetaldehyde to ethanol. An example of this type ofproteins is the AdhE protein in E. coli (Gen Bank No: NP_415757). AdhEappears to be the evolutionary product of a gene fusion. TheNH₂-terminal region of the AdhE protein is highly homologous toaldehyde:NAD⁺ oxidoreductases, whereas the COOH-terminal region ishomologous to a family of Fe²⁺-dependent ethanol:NAD⁺ oxidoreductases(Membrillo-Hernandez et al., (2000) J. Biol. Chem. 275: 33869-33875).The E. coli AdhE is subject to metal-catalyzed oxidation and thereforeoxygen-sensitive (Tamarit et al. (1998) J. Biol. Chem. 273:3027-32).

2) Proteins that catalyse the reversible conversion of acetyl-Coenzyme Ato acetaldehyde in strictly or facultative anaerobic micro-organisms butdo not possess alcohol dehydrogenase activity. An example of this typeof proteins has been reported in Clostridium kluyveri (Smith et al.(1980) Arch. Biochem. Biophys. 203: 663-675). An acetylatingacetaldehyde dehydrogenase has been annotated in the genome ofClostridium kluyveri DSM 555 (GenBank No: EDK33116). A homologousprotein AcdH is identified in the genome of Lactobacillus plantarum(GenBank No: NP_784141). Another example of this type of proteins is thesaid gene product in Clostridium beijerinckii NRRL B593 (Toth et al.(1999) Appl. Environ. Microbiol. 65: 4973-4980, GenBank No: AAD31841).

3) Proteins that are part of a bifunctional aldolase-dehydrogenasecomplex involved in 4-hydroxy-2-ketovalerate catabolism. Suchbifunctional enzymes catalyze the final two steps of the meta-cleavagepathway for catechol, an intermediate in many bacterial species in thedegradation of phenols, toluates, naphthalene, biphenyls and otheraromatic compounds (Powlowski and Shingler (1994) Biodegradation 5,219-236). 4-Hydroxy-2-ketovalerate is first converted by4-hydroxy-2-ketovalerate aldolase to pyruvate and acetaldehyde,subsequently acetaldehyde is converted by acetylating acetaldehydedehydrogenase to acetyl-CoA. An example of this type of acetylatingacetaldehyde dehydrogenase is the DmpF protein in Pseudomonas sp CF600(GenBank No: CAA43226) (Shingler et al. (1992) J. Bacteriol. 174:711-24). The E. coli MphF protein (Ferrandez et al. (1997) J. Bacteriol.179: 2573-2581, GenBank No: NP_414885) is homologous to the DmpF proteinin Pseudomonas sp. CF600.

A suitable nucleic acid sequence may in particular be found in anorganism selected from the group of Escherichia, in particular E. coli;Mycobacterium, in particular Mycobacterium marinum, Mycobacteriumulcerans, Mycobacterium tuberculosis; Carboxydothermus, in particularCarboxydothermus hydrogenaformans; Entamoeba, in particular Entamoebahistolytica; Shigella, in particular Shigella sonnei; Burkholderia, inparticular Burkholderia pseudomallei, Klebsiella, in particularKlebsiella pneumoniae; Azotobacter, in particular Azotobactervinelandii; Azoarcus sp; Cupriavidus, in particular Cupriavidustaiwanensis; Pseudomonas, in particular Pseudomonas sp. CF600;Pelomaculum, in particular Pelotomaculum thermopropionicum. Preferably,the nucleic acid sequence encoding the NAD⁺-dependent acetylatingacetaldehyde dehydrogenase originates from Escherichia, more preferablyfrom E. coli.

Particularly suitable is an mhpF gene from E. coli, or a functionalhomologue thereof. This gene is described in Ferrández et al. (1997) J.Bacteriol. 179:2573-2581. Good results have been obtained with S.cerevisiae, wherein an mhpF gene from E. coli has been incorporated.

In a further advantageous embodiment the nucleic acid sequence encodingan (acetylating) acetaldehyde dehydrogenase is from, in particularPseudomonas, dmpF from Pseudomonas sp. CF600.

In principle, the nucleic acid sequence encoding the NAD⁺-dependent,acetylating acetaldehyde dehydrogenase may be a wild type nucleic acidsequence.

A preferred nucleic acid sequence encodes the NAD⁺-dependent,acetylating acetaldehyde dehydrogenase represented by SEQ ID NO: 2, SEQID NO: 29, or a functional homologue of SEQ ID NO: 2 or SEQ ID NO: 29.In particular the nucleic acid sequence comprises a sequence accordingto SEQ ID NO: 1, SEQ ID NO: 28 or a functional homologue of SEQ ID NO: 1or SEQ ID NO: 28.

Further, an acetylating acetaldehyde dehydrogenase (or nucleic acidsequence encoding such activity) may in for instance be selected fromthe group of Escherichia coli adhE, Entamoeba histolytica adh2,Staphylococcus aureus adhE, Piromyces sp. E2 adhE, Clostridium kluyveriEDK33116, Lactobacillus plantarum acdH, and Pseudomonas putida YP001268189. For sequences of these enzymes, nucleic acid sequencesencoding these enzymes and methodology to incorporate the nucleic acidsequence into a host cell, reference is made to WO 2009/013159, inparticular Example 3, Table 1 (page 26) and the Sequence ID numbersmentioned therein, of which publication Table 1 and the sequencesrepresented by the Sequence ID numbers mentioned in said Table areincorporated herein by reference.

Usually, a cell according to the invention also comprises anacetyl-Coenzyme A synthetase, which enzyme catalyses the formation ofacetyl-coenzyme A from acetate. This enzyme may be present in thewild-type cell, as is for instance the case with S. cerevisiae whichcontains two acetyl-Coenzyme A synthetase isoenzymes encoded by the ACS1[SEQ ID NO: 17] and ACS2 [SEQ ID NO: 18] genes (van den Berg et al(1996) J. Biol. Chem. 271:28953-28959), or a host cell may be providedwith one or more heterologous gene(s) encoding this activity, e.g. theACS1 and/or ACS2 gene of S. cerevisiae or a functional homologue thereofmay be incorporated into a cell lacking acetyl-Coenzyme A synthetaseisoenzyme activity.

Further, in particular in view of an efficient ethanol production, butalso for an efficient NADH oxidation, it is preferred that the cellcomprises an NAD⁺-dependent alcohol dehydrogenase (EC 1.1.1.1). Thisenzyme catalyses the conversion of acetaldehyde into ethanol. The cellmay naturally comprise a gene encoding such a dehydrogenase, as is decase with S. cerevisiae (ADH1-5) [SEQ ID NO: 19-23], see ‘Lutstorf andMegnet. 1968 Arch. Biochem. Biophys. 126:933-944’, or ‘Ciriacy, 1975,Mutat. Res. 29:315-326’), or a host cell may be provided with one ormore heterologous gene(s) encoding this activity, e.g. any or each ofthe ADH1-5 genes of S. cerevisiae or functional homologues thereof maybe incorporated into a cell lacking NAD⁺-dependent alcohol dehydrogenaseactivity.

Specifically preferred cells according to the invention are cells of theS. cerevisiae strain deposited on 16 Jul. 2009 at the Centraalbureauvoor Schimmelcultures (Utrecht, the Netherlands) having deposit numberCBS125049.

In specific aspect, the present invention is directed to a method ofpreparing a recombinant yeast cell according to the invention.

The genetic modification of a cell, comprising the incorporation of oneor more heterologous nucleic acid sequences into a host cell, andusually comprising mutation (including complete deletion) of a geneencoding an enzymatic activity needed for the NADH-dependent glycerolsynthesis, can be based on common general knowledge, e.g. by standardgenetic and molecular biology techniques as generally known in the artand have been previously described (e.g. Maniatis el al. 1982 “Molecularcloning: a laboratory manual”. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.; Miller 1972 “Experiments in molecular genetics”,Cold Spring Harbor Laboratory, Cold Spring Harbor; Sambrook and Russell2001 “Molecular cloning: a laboratory manual” (3rd edition), Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press; F. Ausubel etal., eds., “Current protocols in molecular biology”, Green Publishingand Wiley Interscience, New York 1987).

A method of the invention for preparing a recombinant yeast cellaccording to the invention, comprising:

(a) providing a yeast cell, preferably a yeast cell selected from thegroup of yeast cells lacking enzymatic activity needed for theNADH-dependent glycerol synthesis and yeast cell having a reducedenzymatic activity with respect to glycerol synthesis compared to itscorresponding wild-type yeast cell;

(b) obtaining a nucleic acid segment comprising a gene that isheterologous to said yeast cell and encodes an enzyme that hasNAD⁺-dependent acetylating acetaldehyde dehydrogenase activity, andwherein said gene is operably linked to a promoter functional in saidyeast cell;

(c) if desired (e.g. if the yeast cell lacks acetyl-Coenzyme Asynthetase activity or in which the activity of acetyl-Coenzyme Asynthetase(s) is limiting the overall in vivo activity of the pathwayfor conversion of acetate into ethanol or expresses acetyl-Coenzyme Asynthetase in a cellular compartment that is not compatible with its usein the invention), obtaining a nucleic acid segment comprising a genethat is heterologous to said yeast cell and encodes an enzyme that hasan acetyl-Coenzyme A synthetase activity, and wherein said gene isoperably linked to a promoter functional in said yeast cell;

(d) if desired (e.g. if the yeast cell lacks NAD⁺-dependent alcoholdehydrogenase activity or in which NAD⁺-dependent alcohol dehydrogenaseactivity activity is limiting the in vivo activity of the pathway forconversion of acetate into ethanol or expresses NAD⁺-dependent alcoholdehydrogenase in a cellular compartment that is not compatible with itsuse in the invention), obtaining a nucleic acid segment comprising agene that is heterologous to said yeast cell and encodes an enzyme thathas an NAD⁺-dependent alcohol dehydrogenase activity, and

wherein said gene is operably linked to a promoter functional in saidyeast cell; and

(e) transforming a yeast cell with said nucleic acid segment or segmentsthereby providing a recombinant yeast cell which expresses saidheterologous gene and wherein said recombinant yeast cell exhibitsreduced NADH-dependent glycerol synthesis under fermentative conditionsas compared to a corresponding non-recombinant yeast cell or wherein insaid yeast cell NADH-dependent glycerol synthesis under fermentativeconditions is absent.

Promoters for yeast cells are known in the art and can be, for example,the triosephosphate dehydrogenase TPH promoters,glyceraldehyde-3-phosphate dehydrogenase TDH3 promoters, translationalelongation factor EF-1 alpha TEF1 promoters, the alcohol dehydrogenaseADH1 promoters, glucose-6-phosphate dehydrogenase gpdA and ZWF1promoters, protease promoters such as pepA, pepB, pepC, the glucoamylaseglaA promoters, amylase amyA, amyB promoters, the catalase catR or catApromoters, glucose oxidase goxC promoter, beta-galactosidase lacApromoter, alpha-glucosidase aglA promoter, translation elongation factortefA promoter, xylanase promoters such as xlnA, xlnB, xlnC, xlnD,cellulase promoters such as eglA, eglB, cbhA, promoters oftranscriptional regulators such as areA, creA, xlnR, pacC, prtT, ctc orany other, and can be found among others at the NCBI website (on theWorld Wide Web at ncbi.nlm.nih.gov/entrez/).

In a preferred embodiment, the heterologous nucleic acid sequence to beintroduced into a yeast cell when preparing a recombinant yeast cell ofthe invention, is incorporated into a vector, and the transformation ofthe cell is carried out with the vector. If more than one heterologousnucleic acid sequence (encoding different enzymatic activities ortogether encoding a single enzymatic activity) are to be incorporated,these nucleic acid sequences may be present in a single vector or thesenucleic acid sequences may be incorporated as part of separate vectors.

Accordingly, the invention is further directed to a vector for theexpression of a heterologous polypeptide in a yeast cell, in particulara yeast cell, said expression vector comprising one or more heterologousnucleic acid sequences operably linked to a promoter functional in theyeast cell and said heterologous nucleic acid sequence(s) encoding apolypeptide having enzymatic activity for converting acetyl-Coenzyme Ainto acetaldehyde in (the cytosol of) said yeast cell, wherein saidpolypeptide preferably comprises a sequence according to SEQ ID NO:2 ora functional homologue thereof.

The vector (used in a method of) the invention may be a phage vector, abacterial vector, a centromeric, episomal or integrating plasmid vectoror a viral vector.

In a preferred embodiment the vector is a vector for expression inSaccharomyces, in particular S. cerevisiae. In such embodiment, theheterologous nucleic acid sequence encoding NAD⁺-dependent acetylating,acetaldehyde dehydrogenase activity may be codon(-pair) optimized forexpression in Saccharomyces, in particular S. cerevisiae, although goodresults have been achieved with a wild-type encoding nucleic acidsequence.

In order to achieve optimal expression in a specific cell (such as S.cerevisiae), the codon (pair) usage of the heterologous gene may beoptimized by using any one of a variety of synthetic gene designsoftware packages, for instance GeneOptimizer® from Geneart AG(Regensburg, Germany) for codon usage optimization or codon pair usageoptimization as described in PCT/EP2007/05594. Such adaptation of codonusage ensures that the heterologous genes, which are for instance ofbacterial origin, are effectively processed by the yeast transcriptionand translation machinery. Optimisation of codon pair usage will resultin enhanced protein expression in the yeast cell. The optimizedsequences may for instance be cloned into a high copy yeast expressionplasmid, operably linked to a (preferably constitutive) promoterfunctional in a fungus (yeast).

As indicated above, the invention is further directed to the preparationof ethanol.

For a method of preparing ethanol, a cell according to the invention isused which under anaerobic conditions ferments a sugar thereby formingethanol Suitable yeast cells for fermenting the sugar are generallyknown and include amongst others S. cerevisiae. In a method of theinvention, a yeast cell is used that also produces an NAD⁺-dependentacetylating acetaldehyde dehydrogenase (EC 1.2.1.10). This cell can beobtained as described above, and illustrated in the example hereinbelow. If used for the preparation of ethanol, the cell preferably alsoincludes an acetyl-Coenzyme A synthetase activity. If used for thepreparation of ethanol, the cell preferably also includes anNAD⁺-dependent alcohol dehydrogenase activity. These activities may benaturally present, as in S. cerevisiae, or provided by geneticmodification (see also herein above).

The fermentation conditions can in principle be based on generally knownconditions, e.g., as described in the review by Van Maris cited above,or the references cited therein, with the proviso that typically themedium wherein the fermentation is carried comprises acetate in additionto the fermentable carbohydrate(s).

The molar ratio acetate to carbohydrate consumed by anaerobic culturesof the yeast cells modified according to the invention is usually atleast 0.004, in particular at least 0.01, at least 0.03, at least 0.1,or at least 0.2. The molar ratio acetate to carbohydrate present inhydrolysates of lignocellulosic biomass is usually less than 0.70, inparticular 0.5 or less, more in particular 0.3 or less, or 0.2 or less.Herein the number of moles of carbohydrate is based on monosaccharideunits, i.e. one mole of a oligo/polysaccharide having n monosaccharideunits counts as n mol.

In absolute terms, the fermentable carbohydrate concentration is usuallyin the range of 65 to 400 g/L, in particular in the range of 100 to 250g/L.

In absolute terms, the acetate concentration is usually in the range of0.5 to 20 g/L, in particular in the range of 1-15 g/L, more inparticular in the range of 2 to 12 g/L.

The pH can be chosen dependent on what is acceptable to the organismthat is used, based on common general knowledge or can be routinelydetermined. Usually the fermentation is carried out at a neutral oracidic pH, in particular at a pH in the range of 2-7, more in particularat a pH of 3-6, even more in particular 3.5-5.5 (apparent pH as measuredin the fermentation medium at the temperature at which fermentationtakes place).

The temperature can be chosen dependent on what is acceptable to theorganism that is used. Usually the temperature is in the range of 15-50degrees C., in particular in the range of 25-45 degrees C.

As a fermentable carbohydrate in principle any carbohydrate can be usedthat is metabolized by the specific recombinant cell, with ethanol as ametabolic product. The cell may naturally comprise the requiredmetabolic enzyme system or the cell may have been genetically modifiedto that purpose, e.g. as described by the review by Van Maris, thereferences cited therein, or as described in the present disclosure.Preferably, the fermentable carbohydrates in the hydrolysate comprise atleast one carbohydrate selected from the group of hexoses, pentoses andoligosaccharides comprising one or more hexose and/or pentose units. Inparticular in case the recombinant cell is from the group Saccharomyces,preferably S. cerevisiae at least one carbohydrate selected fromglucose, fructose, sucrose, maltose, galactose, xylose, arabinose andmannose is used. Good results have been obtained with glucose.

A method according to the invention is in particular suitable forpreparing ethanol using a hydrolysate of at least one polymer selectedfrom cellulose, hemicellulose and pectin, preferably at least onepolymer selected from hemicellulose and pectin, because upon hydrolysisof these polymers acetate is typically released by hydrolysis or formedas a breakdown product. In particular, the hydrolysate may be hydrolysedlignocellulosic material, such as lignocellulosic biomass. For instance,lignocellulosic material may be selected from agricultural material, forinstance cotton, straw, (elephant) grass, bagasse, corn stover,lignocellosic aquatic plant material, sugar beet pulp, citrus peels,lignocellulosic materials from forestry, such as lignocellulosicwaste-materials from trees or bushes (trimmed/lopped/pruned plantmaterial, saw dust, etc.) or trees or bushes specifically grown as asource for lignocellulosic materials, e.g. poplar trees,(ligno)cellulosic waste from industry, e.g. wood-pulp, waste-paper.

Preferably, a lignocellulosic hydrolysate comprises one or morefermentable sugars (notably glucose, xylose and arabinose) and acetate,which have formed during hydrolysis. Thus, in a preferred embodiment ofthe invention the preparation of ethanol comprises a step wherein alignocellulosic material is hydrolysed, thereby forming a hydrolysatecomprising one or more fermentable carbohydrates, in particular glucoseand optionally one or more other hexoses and pentoses, and acetate,which hydrolysate is thereafter contacted with a recombinant cell of theinvention. The relative concentrations of acetate and fermentablecarbohydrates in the substrate that is contacted with the recombinantcell of the invention can be modified by optimizing conditions forhydrolysis, by blending different hydrolysates and/or by blending with(partially) refined sources of carbohydrates and/or acetic acid.

Suitable hydrolysis methodology may be based on the Van Maris reviewcited above or the references cited therein, and include enzymatichydrolysis, thermal hydrolysis, chemical hydrolysis and combinationsthereof. The polymers are usually hydrolysed to the extent that at least50%, preferably at least 90%, in particular at least 95% of the chainsare degraded to monosaccharide units or to monosaccharide units anddisaccharide units.

The invention is now illustrated by the following example:

EXAMPLES Example 1 Materials and Methods

Strain Construction and Maintenance

The Saccharomyces cerevisiae strains used (Table 1) originate from theCEN.PK family, which was previously identified as a suitable backgroundfor combined genetic and physiological studies (van Dijken et al. (2000)Enzyme Microb. Technol. 26:706-714).

TABLE I Saccharomyces cerevisiae strains used Strain Relevant genotypeSource/reference CEN.PK113-5D MATa ura3 GPD1 GPD2 EUROSCARF straincollection, Frankfurt, Germany IME076 MATa ura3 GPD1 GPD2 (reference)p426_GPD(URA3) CEN.PK102-3A MATa ura3 leu2 GPD1 GPD2 EUROSCARF straincollection, Frankfurt, Germany RWB0094 MATa ura3 leu2gpd1(−1.1133)::loxP- BIRD Engineering, KanMX-loxP gpd2(−2.1281)::hphMX4Rotterdam IMZ008 MATa ura3 leu2 gpd1(−1.1133)::loxPgpd2(−2.1281)::hphMX4 YEplac181(LEU2) IMZ132 MATa ura3 leu2gpd1(−1.1133)::loxP Deposited at (CBS125049) gpd2(−2.1281)::hphMX4YEplac181(LEU2) Centraalbureau pUDE43(URA3 pTHD3::mhpF voor (E.coli)::CYC1t) Schimmelcultures on Jul. 16, 2009 IMZ127 MATa ura3 leu2gpd1(−1.1133)::loxP gpd2Δ(−2.1281)::hphMX4 YEplac181(LEU2)p426_GPD(URA3)

To disrupt the GPD1 gene (YDL022W), the loxP-KanMX-loxP disruptioncassette may be amplified by PCR according to Güldener et al. (1996,Nucleic Acids Res. 24:2519-2524), using a primer set containing45-nucleotide flanking regions homologous to sequences within the GPD1gene and approximately 20-nucleotide homologous to sequences of thedisruption module of pUG6 (Güldener et al. (1996) Nucleic Acids Res.24:2519-2524) (FIG. 2, Table 2). Similarly, plasmid pAG32 ((Goldsteinand McCusker (1999) Yeast 15:1541-1553) may be used as a template forPCR amplification of the hphMX4 disruption module. For construction of aGPD2 (YOL059W) disruption cassette a primer set may be used containing45-nucleotide flanking regions homologous to sequences within the GPD2gene and 20-nucleic acid sequences homologous to sequences of thedisruption module of pAG32 (Table 2).

TABLE 2 Oligonucleotides for inactivation of the GPD1 and GPD2genes and for verification of correct disruption bydiagnostic PCR. Gene disruption oligonucleotides;nucleotides homologous to the sequence to eitherthe left (5′ side) or right (3′ side) of the genesto be deleted are indicated in capitals; lower caseletters indicate nucleotides homologous to thesequence of the disruption cassettes. Target gene GPD1/YDL022WGPD2/YOL059W Gene fw fw disruption 5′TTGTACACCCCCCCCCTCCACA5′TCAATTCTCTTTCCCTTTCCTTT primers AACACAAATATTGATAATATAAAcaTCCTTCGCTCCCCTTCCTTATC gctgaagcttcgtacgc ccaggctgaagcttcgtacg[SEQ ID NO: 5] [SEQ ID NO: 7] rv rv 5′AATCTAATCTTCATGTAGATCTA5′GTGTCTATTCGTCATCGATGTCT ATTCTTCAATCATGTCCGGCGgcataAGCTCTTCAATCATCTCCGGTAGg ggccactagtggatctg cataggccactagtggatc[SEQ ID NO: 6] [SEQ ID NO: 8] Verification fw 5′ GPD1 fw 5′ GPD2primers- 5′ CCCACCCACACCACCAATAC 5′ GTTCAGCAGCTCTTCTCTAC target gene[SEQ ID NO: 9] [SEQ ID NO: 11] specific rv 3′ GPD1 rv 3′ GPD15′ CGGACGCCAGATGCTAGAAG 5′ CCAAATGCGACATGAGTCAC [SEQ ID NO: 10][SEQ ID NO: 12] Verification fw KANB fw KANB primers-5′ CGCACGTCAAGACTGTCAAG 5′ CGCACGTCAAGACTGTCAAG disruption[SEQ ID NO: 13] [SEQ ID NO: 15] cassette rv KANA rv KANA specific5′ TCGTATGTGAATGCTGGTCG 5′ TCGTATGTGAATGCTGGTCG [SEQ ID NO: 14][SEQ ID NO: 16]

Transformation of the PCR amplified GPD1 and GPD2 disruption cassettesto Saccharomyces cerevisiae strain CEN.PK102-3A (Table 1) may beperformed according to the protocols described by Güldener et al(Nucleic Acids Res. (1996) 24:2519-2524), followed by selection oftransformants on YPD complex medium (Burke et al. (2000) Methods inyeast genetics, Cold Spring Harbour Press Plainview, N.Y.) with 200 mg/LG-418 for strains transformed with the KanMX disruption cassette and 300mg/L hygromycin B for strains transformed with the hphMX4 disruptioncassette. Confirmation of correct integration of the GPD1 genedisruption cassette may be checked by colony PCR, using combinations ofprimer sets 5′ GPD1\KANA and 3′GPD1\KANB (Table 2). The correctinactivation of GPD2 can be similarly verified with primer sets 5′GPD2\KANA and 3′GPD2\KANB (Table 2). Strain RWB0094, carrying deletionsin the open reading frames of the GPD1 and GPD2 genes of strainCEN.PK102-3A (MATa ura3 leu2) were replaced by the loxP-KanMX-loxPcassette and the hphMX4 cassette, respectively, was acquired from BIRDEngineering, Rotterdam, The Netherlands. The KanMX marker of strainRWB0094 was removed by expression of the Cre recombinase (Güldener et al(1996) Nucleic Acids Res. 24:2519-2524) and its leucine auxotrophy wascomplemented by transformation with the LEU2-bearing plasmid YBPlac181(Gietz, R. D. and S. Akio. (1988) Gene 74:527-534), yielding strainIMZ008. FIG. 1 2 schematically shows the gene disruption procedure,replacement of the GPD1 ORF by KanMX and GPD2 ORF by hphMX4. Arrowsindicate oligonucleotide primers for verification by diagnostic PCR ofcorrect gene inactivation.

Transformation of strain IMZ008 with the URA3-bearing mhpF expressionplasmid pUDE43 (see below) yielded the prototrophic, mhpF-expressingstrain IMZ132, transformation with the URA3-bearing ‘empty’ vectorp426_GPD yielded strain IMZ127. Finally, transformation of strainCEN.PK113-5D (ura3) with p426_GPD yielded the prototrophic GPD1 GPD2reference strain IME076. Cultures transformed with deletion cassetteswere plated on YPD complex medium containing G418 (200 mg l⁻¹) orhygromycin (200 mg l⁻¹). Successful integration of the deletioncassettes was confirmed by diagnostic PCR.

Stock cultures of all strains were grown in shake flasks containing 100ml of synthetic medium (see below) with 20 g l⁻¹ glucose as the carbonsource. After adding 30% (v/v) glycerol, 1-ml aliquots of stationaryphase cultures were stored at −80° C.

Plasmid Construction

The E. coli mhpF gene (EMBL accession number Y09555.7) was PCR amplifiedfrom E. coli K12 strain JM109 genomic DNA using primer pairs mhpF-FW(5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAGTAAGCGTAAAGTCGCCATTATCGG-3′ [SEQID NO: 3]) and mhpF RV (5′ GGGGACCACTTTGTACAAGAAAGCTGGGTGTTCATGCCGCTTCTCCTGCCTTGC-3′, [SEQ ID NO: 4]), which containedattB1 and attB2 sequences, respectively. The polymerase chain reaction(PCR) was performed using Phusion® Hot Start High-Fidelity DNAPolymerase (Finnzymes Oy, Espoo, Finland) according to manufacturerspecifications and in a Biometra TGradient Thermocycler (Biometra,Göttingen, Germany) with the following settings: 25 cycles of 10 sdenaturation at 98° C. and 30 s annealing and extension at 72° C. The1011 bp PCR product was cloned using Gateway® cloning technology(Invitrogen, Carlsbad, Calif., USA). Plasmid pDONR221, using the BPreaction, was used to create the entry clone, designated as plasmidpUD64. From this entry clone and the multicopy plasmid pAG426GPD-ccdB(Addgene, Cambridge, Mass., USA) the yeast expression plasmid pUDE43,was constructed employing the LR reaction. Transformations ofrecombination reaction products into competent E. coli K12 strain JM109were performed according to the Z-Competent™ E. coli Transformation Kit(Zymoresearch Corporation, Orange, USA) and plated on LB mediacontaining either ampicillin (100 mg l⁻¹) or kanamycin (50 mg·l⁻¹).Yeast transformations were performed according to Burke et al. (Methodsin yeast genetics (2000.) Cold Spring Harbor Laboratory Press Plainview,N.Y.) After transformations with the yeast expression plasmid, cellswere plated on synthetic media. Successful insertion of multicopyplasmid pUDE43 was confirmed by diagnostic PCR using the primer pairsfor cloning.

Cultivation and Media

Shake-flask cultivation was performed at 30° C. in a synthetic medium(46). The pH of the medium was adjusted to 6.0 with 2 M KOH prior tosterilization. Precultures were prepared by inoculating 100 ml mediumcontaining 20 g l⁻¹ glucose in a 500 mL shake-flask with (1 ml) frozenstock culture. After 24 h incubation at 30° C. in an Innova® incubatorshaker (200 rpm, New Brunswick Scientific, NJ, USA), cultures weretransferred to bioreactors.

Anaerobic batch fermentations were carried out at 30° C. in 2-litrelaboratory bioreactors (Applikon, Schiedam, The Netherlands) with aworking volume of 1 litre. Synthetic medium with 20 g l⁻¹ glucose (46)was used for all fermentations and supplemented with 100 μl l⁻¹ ofsilicone antifoam (Silcolapse 5020, Caldic Belgium, Bleustar Silicones)as well as with the anaerobic growth factors, ergosterol (0.01 g l⁻¹)and Tween 80 (0.42 g l⁻¹) dissolved in ethanol. This resulted in 11-13mM ethanol in the medium. Where indicated, acetic acid was added at aconcentration of 2 g l⁻¹ and the pH was readjusted to 5.0 prior toinoculation. Culture pH was maintained at 5.0 by the automatic additionof 2M KOH. Cultures were stirred at 800 rpm and sparged with 0.5 L min⁻¹nitrogen (<10 ppm oxygen). Dissolved oxygen was monitored with anautoclavable oxygen electrode (Applisens, Schiedam, The Netherlands). Tominimize diffusion of oxygen, bioreactors were equipped with Norprenetubing (Cole Palmer Instrument Company, Vernon Hills, USA). Allfermentations were earned out at least in duplicate.

Determination of Culture Dry Weight and Optical Density

Culture samples (10 ml) at selected time intervals were filtered overpre-weighed nitrocellulose filters (pore size 0.45 μm; GelmanLaboratory, Ann Arbor, USA). After removal of medium the filters werewashed with demineralized water and dried in a microwave oven (Bosch,Stuttgart, Germany) for 20 min at 350 W and weighed. Duplicatedeterminations varied by less than 1%. Culture growth was also monitoredvia optical density readings at a wavelength of 660 nm on a Novaspec® IIspectrophotometer.

Gas Analysis

Exhaust gas was cooled in a condenser (2° C.) and dried with a Permapuredryer type MD-110-48P-4 (Permapure, Toms River, USA). Oxygen and carbondioxide concentrations were determined with a NGA 2000 analyzer(Rosemount Analytical, Orrville, USA). Exhaust gas-flow rate and carbondioxide production rates were determined as described previously (3). Incalculating these biomass-specific rates, a correction was made forvolume changes caused by withdrawing culture samples.

Metabolite Analysis

Supernatant obtained by centrifugation of culture samples was analyzedfor glucose, acetic acid, succinic acid, lactic acid, glycerol andethanol via HPLC analysis on a Waters Alliance 2690 HPLC (Waters,Milford, USA) containing a Biorad HPX 87H column (Biorad, Hercules,USA). The column was eluted at 60° C. with 0.5 g l⁻¹ H₂SO₄ at a flowrate of 0.6 ml min⁻¹. Detection was by means of a Waters 2410refractive-index detector and a Waters 2487 UV detector. Initial andfinal glycerol concentrations were further determined using an enzymaticdetermination kit (R-Biopharm AG, Darmstadt, Germany). Duringcultivation in bioreactors that are sparged with nitrogen gas, asignificant fraction of the ethanol is lost through the off gas. Tocorrect for this, ethanol evaporation kinetics were analyzed inbioreactors operated under identical conditions at different workingvolumes with sterile synthetic medium. The resulting volume-dependentethanol evaporation constants (for this set-up equal to 0.0080 dividedby the volume in litres, expressed in h⁻¹) were used to correct HPLCmeasurements of ethanol concentrations in culture supernatants, takinginto account changes in volume that were caused by sampling.

Enzyme Activity Assays

Cell extracts for activity assays of NAD⁺-dependent acetaldehydedehydrogenase (acetylating) were prepared from exponentially growinganaerobic batch cultures as described previously (Abbott et al. Appl.Environ. Microbiol. 75:2320-2325). NAD⁺-dependent acetaldehydedehydrogenase (acetylating) activity was measured at 30° C. bymonitoring the oxidation of NADH at 340 nm. The reaction mixture (totalvolume 1 ml) contained 50 mM potassium phosphate buffer (pH 7.5), 15 mMNADH and cell extract. The reaction was started by addition of 0.5 mMacetyl-Coenzyme A. For glycerol 3-phosphate dehydrogenase (EC 1.1.1.8)activity determination, cell extracts were prepared as described aboveexcept that the phosphate buffer was replaced by triethanolamine buffer(10 mM, pH 5) (5,19). Glycerol-3-phosphate dehydrogenase activities wereassayed in cell extracts at 30° C. as described previously (Blomberg andAdler (1989), J. Bacteriol. 171:1087-1092. Reaction rates wereproportional to the amounts of cell extract added. Proteinconcentrations were determined by the Lowry method (Lowry et al (1951)J. Biol. Chem. 193:265-275) using bovine serum albumin as a standard.

Results

Growth and Product Formation in Anaerobic Batch Cultures

When cultures of the prototrophic reference strain S. cerevisiae IME076(GPD1 GPD2) were supplemented with 2.0 g l⁻¹ acetic acid, the specificgrowth rate (0.32 h⁻¹) was identical to that reported for cultures grownin the absence of acetic acid (0.34 h⁻¹), Kuyper et al. (2005) FEMSYeast Res. 5:399-409. The addition of acetic acid led to a slightdecrease of the biomass yield and, consequently a decrease of theglycerol yield on glucose relative to cultures grown in the absence ofacetic acid (FIG. 2, Table 3).

This effect has been attributed to the higher rate of glucosedissimilation for intracellular pH homeostasis due to diffusion ofacetic acid into the cell, which in turn results in a lower biomassyield on glucose. Under the same conditions, an isogenic gpd1Δ gpd2Δstrain, in which absence of NAD⁺-dependent glycerol-3-phosphatedehydrogenase activity was confirmed in cell extracts (Table 2), wascompletely unable to grow anaerobically (FIG. 2), consistent with thenotion that glycerol production via Gpd1 and Gpd2 is essential for NADHreoxidation in anaerobic cultures of S. cerevisiae.

TABLE 3 Physiology of the engineered S. cerevisiae strain IMZ132 and theempty-vector reference strain IME076 during anaerobic batch cultivationon synthetic medium (pH 5) with glucose-acetate mixtures Yeast strainIME076 IMZ132 Relevant genotype GPD1 GPD2 gpd1Δ gpd2Δ + mhpF Glycerol3-phosphate 0.034 ± 0.003 <0.002 dehydrogenase (μmol mg protein⁻¹ min⁻¹)Acetaldehyde dehydrogenase <0.002 0.020 ± 0.004 (acetylating) (μmol mgprotein⁻¹ min⁻¹) Specific growth rate (h⁻¹) 0.32 ± 0.01 0.14 ± 0.01Biomass yield on glucose (g g⁻¹) 0.083 ± 0.000 0.082 ± 0.009 Biomassyield on acetate (g g⁻¹) n.a. 3.8 ± 0.5 Glycerol yield on glucose (gg⁻¹) 0.073 ± 0.007 <0.002 Ethanol yield on glucose (g g⁻¹) 0.39 ± 0.010.43 ± 0.01 Not corrected for evaporation Ethanol yield on glucose (gg⁻¹) 0.41 ± 0.01 0.47 ± 0.01 Corrected for evaporation n.a., notapplicable.

Expression of the E. coli mhpF gene in a gpd1Δ gpd2Δ strain, resultingin acetyl-CoA dependent rates of NADH reduction in cell extracts of0.020 μmol min⁻¹ (mg protein)⁻¹ (Table 3), did not enable anaerobicgrowth when glucose was the sole carbon source. However, when the mediumwas supplemented with 2.0 g l⁻¹ acetic acid, exponential growth wasobserved at a specific growth rate of 0.14 h⁻¹. No formation of glyceroloccurred during cultivation. (FIG. 2, Table 3). The trace amounts (<1mM) of glycerol present in cultures of gpd1Δ gpd2Δ strains originatefrom the inoculum cultures which were started from frozen glycerolstocks. Ethanol was the major organic product and the small amounts ofsuccinate and lactate produced were similar to those observed incultures of the reference strain grown under the same conditions (datanot shown).

It is observed that the IMZ132 fermentations (40 h) lasted longer thanthe wild type strain (15 h) and the anaerobic batch cultures weresparged with nitrogen gas. Accordingly, the fraction of ethanol lostthrough evaporation was higher for strain IMZ132. After determination ofthe kinetics of ethanol evaporation in sterile control experiments andcorrection of the ethanol yields, a 13% higher apparent ethanol yield onglucose was shown for the engineered strain according to the invention,using the linear pathway for NADH dependent reduction of acetic acid toethanol (Table 3).

Discussion

The present study provides a proof of principle that,stoichiometrically, the role of glycerol as a redox sink for anaerobicgrowth of S. cerevisiae can be fully replaced by a linear pathway forNADH-dependent reduction of acetate to ethanol. This offers interestingperspectives for large-scale ethanol production from feedstocks thatcontain acetic acid, such as lignocellulosic hydrolysates.

In addition to reducing the organic carbon content of spent media andincreasing the ethanol yield, the reduction of acetic acid to ethanolmay at least partially alleviate acetate inhibition of yeast growth andmetabolism, which is especially problematic at low pH and during theconsumption of pentose sugars by engineered yeast strains

Example 2

This example relates to the fermentative glycerol-free ethanolproduction by a gpd1Δ gpd2Δ Saccharomyces. cerevisiae strain expressinga codon optimized version (SEQ ID NO: 28) of the wild type dmpF genefrom Pseudomonas sp. CF600 (GenBank No: CAA43226) (Shingler et al.(1992) J. Bacteriol. 174:71 1-24).

For this purpose, the same procedures and techniques used for theconstruction and evaluation of the performance of the strain IMZ132(gpd1Δ gpd2Δ mhpF) were used. These procedures and techniques aredescribed in Materials and Methods section of Example 1. In this work,transformation of strain IMZ008 (gpd1Δ gpd2Δ ura3Δ) with theURA3-bearing dmpF expression plasmid pUDE47 yielded the prototrophic,dmpF-expressing strain IMZ130. For the construction of plasmid pUDE47, acodon optimized copy of the dmpF gene (EMBL accession number X60835.1)from Pseudomonas sp. CF600 was ligated into p426_GPD plasmid. Codonoptimization for the expression in S. cerevisiae and ligation into theplasmid p426_GPD were performed by BaseClear BV (Leiden, TheNetherlands). Successful insertion of multicopy plasmid pUDE47 wasconfirmed using diagnostic colony PCR using the primer pairs dmpF-FW(CATTGATTGCGCCATACG (SEQ ID NO:30)) and dmpF-RV (CCGGTAATATCGGAACAGAC(SEQ ID NO: 31)).

Results

Growth and Product Formation in Anaerobic Batch Cultures

Expression of the Pseudomonas sp. CF600 dmpF gene in a gpd1Δ gpd2Δ S.cerevisiae strain gave similar results to the obtained for theexpression of E. coli mhpF gene in the same strain. In anaerobic batchfermentations, similar to strain IMZ132 (gpd1Δ gpd2Δ mhpF), functionalexpression of dmpF gene together with supplementation of the medium with2.0 g l⁻¹ acetic acid, resulted in exponential growth with a specificgrowth rate of 0.11 h⁻¹ (FIG. 1). Strain IMZ130 (gpd1Δ gpd2Δ dmpF) had aslightly longer batch time (55 h) than strain IMZ132 (40 h). Duringcultivation, initial concentration of 20 g l⁻¹ of glucose was completelyconsumed, while no glycerol formation was observed. At the same time,acetate was consumed from 2.1 g l⁻¹ initial concentration to 1.6 g l⁻¹final concentration. Ethanol was the major organic product, exhibitingan ethanol yield on glucose of 0.48 g g⁻¹ (yield corrected by ethanolevaporation). Small amounts of succinate and lactate were produced,similar to those observed in cultures of the reference strain grownunder the same conditions.

The insertion of a synthetic codon optimized copy of the gene dmpF fromPseudomonas sp. CF600, provides another example that it isstoichiometrically possible to substitute glycerol formation as a redoxsink in anaerobic growth of S. cerevisiae by a linear metabolic pathwayfor NADH-dependent reduction of acetate to ethanol. Also this exampleshows that the insertion of (acetalyting) acetaldehyde dehydrogenase ina gpdΔ1 gpd2Δ S. cerevisiae strain resulted in higher ethanol yields onglucose, no formation of by-product glycerol, and the consumption offermentation inhibitor compound acetate.

Sequences E. coli mhpF gene Acetaldehyde dehydrogenase, acylatingSEQ ID NO: 1 ATGAGTAAGCGTAAAGTCGCCATTATCGGTTCTGGCAACATTGGTACCGATCTGATGATTAAAATTTTGCGTCACGGTCAGCATCTGGAGATGGCGGTGATGGTTGGCATTGATCCTCAGTCCGACGGTCTGGCGCGCGCCAGACGTATGGGCGTCGCCACCACCCATGAAGGGGTGATCGGACTGATGAACATGCCTGAATTTGCTGATATCGACATTGTATTTGATGCGACCAGCGCCGGTGCTCATGTGAAAAACGATGCCGCTTTACGCGAAGCGAAACCGGATATTCGCTTAATTGACCTGACGCCTGCTGCCATCGGCCCTTACTGCGTGCCGGTGGTTAACCTCGAGGCGAACGTCGATCAACTGAACGTCAACATGGTCACCTGCGGCGGCCAGGCCACCATTCCAATGGTGGCGGCAGTTTCACGCGTGGCGCGTGTTCATTACGCCGAAATTATCGCTTCTATCGCCAGTAAATCTGCCGGACCTGGCACGCGTGCCAATATCGATGAATTTACGGAAACCACTTCCCGAGCCATTGAAGTGGTGGGCGGCGCGGCAAAAGGGAAGGCGATTATTGTGCTTAACCCAGCAGAGCCACCGTTGATGATGCGTGACACGGTGTATGTATTGAGCGACGAAGCTTCACAAGATGATATCGAAGCCTCAATCAATGAAATGGCTGAGGCGGTGCAGGCTACGTACCGGGTTATCGCCTGAAACAGCGCGTGCAGTTTGAAGTTATCCCGCAGGATAAACCGGTCAATTTACCGGGCGTGGGGCAATTCTCCGGACTGSSSSCAGCGGTCTGGCTGCAAGTCGAAGGCGCAGCGCATTATCTGCCTGCCTATGCGGGCAACCTCGACATTATGACTTCCAGTGCGCTGGCGACAGCGGAAAAAATCGCCCAGTCACTGGCGCGCAAGGCAGGAGAAGCGGCATGAE. coli Acetaldehyde dehydrogenase OS = Escherichia coli(strain K12) GN = mhpF PE = SV = 1 SEQ ID NO: 2MSKRKVAIIGSGNIGTDLMIKILRHGQHLEMAVMVGIDPQSDGLARRMGVATTHEGVIGLMNMPEFADIDIVFDATSAGAHVKNDAALREAKDIRLIDLTPAAIGPYCVPVVNLEANVDQLNVNMVTCGGQATIPMVAAVSRVARVHYAEIIASIASKSAGPGTRANIDEFTETTSRAIEVVGGAAKGKAIIVLNPAEPPLMMRDTVYVLSDEASQDDIEASINEMAEAVQAYVPGYRLKQRVQFEVIPQDKPVNLPGVGQFSGLKTAVWLEVEGAAHYLPAYAGNLDIMTSSALATAEKMAQSLARKAGEAA primer mhpF-FW SEQ ID NO: 3GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAGTAAGCGTAAAGTCGCCATTAT CGGprimer mhpF-RV SEQ ID NO: 4GGGGACCACTTTGTACAAGAAAGCTGGGTGTTCATGCCGCTTCTCCTGCCTTGCGPD1/YDL022W fw gene disruption primer SEQ ID NO: 5TTGTACACCCCCCCCCTCCACAAACACAAATATTGATAATATAAAcagctgaagcttcgta cgcGPD1/YDL022W rv gene disruption primer SEQ ID NO: 6AATCTAATCTTCATGTAGATCTAATTCTTCAATCATGTCCGGCGgcataggccactagtgg atctgGPD2/YOL059W fw gene disruption primer SEQ ID NO: 7TTCAATTCTCTTTCCCTTTCCTTTTCCTTCGCTCCCCTTCCTTATC ccaggctgaagcttcgtacgGPD2/YOL059W rv gene disruption primer SEQ ID NO: 8GTGTCTATTCGTCATCGATGTCTAGCTCTTCAATCATCTCCGGTACgcataggccactag tggatcGPD1/YDL022W fw verification primer SEQ ID NO: 9 CCCACCCACACCACCAATACGPD1/YDL022Wrv verification primer SEQ ID NO: 10 CGGACGCCAGATGCTAGAAGGRD2/YOL059W fw Verification primer SEQ ID NO: 11 GTTCAGCAGCTCTTCTCTACGPD2/YOL059W rv verification primer SEQ ID NO: 12 CCAAATGCGACATGAGTCACGPD1/YDL022W fw disruption cassette specific verification primerSEQ ID NO: 13 CGCACGTCAAGACTGTCAAGGPD1/YDL022W rv disruption cassette specific verification primerSEQ ID NO: 14 TCGTATGTGAATGCTGCTCGGPD2/YOL059W fw disruption cassette specific verification primerSEQ ID NO: 15  CGCACGTCAAGACTGTCAAGGPD2/YOL059W rv disruption cassette specific verification primerSEQ ID NO: 16  TCGTATGTGAATGCTGGTCG S. cerevisiae ACS 1 geneSEQ ID NO: 17 ATGTCGCCCTCTGCCGTACAATCATCAAAACTAGAAGAACAGTCAAGTGAAATTGACAAG TTGAAAGCAAAAATGTCCCAGTCTGCCGCCACTGCGCAGCAGAAGAAGGAACATG AGTATGAACATTTCACTTCGCTCAAGATCGTGCCACAACGGCCCATCTCAGATAGACTGC AGCCCGCAATTGCTACCCACTATTCTCCACACTTGGACGGGTTGCAGGACTATCAGCGCT TGCACAAGGAGTCTATTGAAGACCCTGCTAAGTTCTTCGGTTCTAAAGCTACCCAATTTTT AAACTGGTCTAAGCCATTCGATAAGGTGTTCATCCCAGACCCTAAAACGGGCAGGCCCT CCTTCCAGAACAATGCATGGTTCCTCAACGGCCAATTAAACCCCTGTTACAACTGTGTTG ACAGACATGCCTTGAAGACTCCTAACAAGAAAGCCATTATTTTCGAAGGTGACGAGCCTG GCCAAGGCTATTCCATTACCTACAAGGAACTACTTGAAGAAGTTTGTCAAGTGGCACAAG TGCTGACTTACTCTATGGGCGTTCGCAAGGGCGATACTCTTGCCGTGTACATGCCTATGG TCCCAGAAGCAATCATAACCTTGTTGGCCATTTCCCGTATCGGTGCCATTCACTCCGTAG TCTTTGCCGGGTTTTCTTCCAACTCCTTGAGAGATCGTATCAACGATGGGGACTCTAAAG TTGTCATCACTACAGATGAATCCAACAGAGGTGGTAAAGTCATTGAGACTAAAAGAATTG TTGATGACGCGCTAAGAGAGACCCCAGGCGTGAGACACGTCTTGGTTTATAGAAAGACC AACAATCCATCTGTTGCTTTCCATGCCCCCAGAGATTTGGATTGGGCAACAGAAAAGAAGA AATACAAGACCTACTATCCATGCACACCCGTTGATTCTGAGGATCCATTATTCTTGTTGTA TACGTCTGGTTCTACTGGTGCCCCCAAGGGTGTTCAACATTCTACCGCAGGTTACTTGC TGGGAGCTTTGTTGACCATGCGCTACACTTTTGACACTCACCAAGAAGACGTTTTCTTCAC AGCTGGAGACATTGGCTGGATTACAGGCCACACTTATGTGGTTTATGGTCCCTTACTAT ATGGTTGTGCCACTTTGGTCTTTGAAGGGACTCCTGCGTACCCAAATTACTCCCGTTATT GGGATATTATTGATGAACACAAAGTCACCCAATTTTATGTTGCGCCAACTGCTTTGCGTTT GTTGAAAAGAGCTGGTGATTCCTACATCGAAAATCATTCCTTAAAATCTTTGCGTTGCTT GGGTTCGGTCGGTGAGCCAATTGCTGCTCAAGTTTGGGAGTGGTACTCTGAAAAAATAG GTAAAAATGAAATCCCCATTGTAGACACCTACTGGCAAACAGAATCTGGTTCGCATCTGG TCACCCCGCTGGCTGGTGGTGTTACACCAATGAAACCGGGTTCTGCCTCATTCCCCTTCT TCGGTATTGATGCAGTTGTTCTTGACCCTAACACTGGTGAAGAACTTAACACCAGCCACG CAGAGGGTGTCCTTGCCGTCAAAGCTGCATGGCCATCATTTGCAAGAACTATTTGGAAAA ATCATGATAGGTATCTAGACACTTATTTGAACCCTTACCCTGGCTACTATTTCACTGGTGA TGGTGCTGCAAAGGATAAGGATGGTTATATCTGGATTTTGGGTCGTGTAGACGATGTGG TGAACGTCTCTGGTCACCGTCTGTCTACCGCTGAAATTGAGGCTGCTATTATCGAAGATC CAATTGTGGCCGAGTGTGCTGTTGTCGGATTCAACGATGACTTGACTGGTCAAGCAGTTG CTGCATTTGTGGTGTTGAAAAACAAATCTAGTTGGTCCACCGCAACAGATGATGAATTAC AAGATATCAAGAAGCATTTGGTCTTTACTGTTAGAAAAGACATCGGGCCATTTGCCGCAC CAAAATTGATCATTTTAGTGGATGACTTGCCCAAGACAAGATCCGGCAAAATTATGAGAC GTATTTTAAGAAAAATCCTAGCAGGAGAAAGTGACCAACTAGGCGACGTTTCTACATTGT CAAACCCTGGCATTGTTAGACATCTAATTGATTCGGTCAAGTTGTAA S. cerevisiae ACS 2 geneSEQ ID NO: 18 ATGACAATCAAGGAACATAAAGTAGTTTATGAAGCTCACAACGTAAAGGCTCTTAAGGCT CCTCAACATTTTTACAACAGCCAACCCGGCAAGGGTTACGTTACTGATATGCAAC ATTATCAAGAAATGTATCAACAATCTATCAATGAGCCAGAAAAATTCTTTGATAAGATGG CTAAGGAATACTTGCATTGGGATGCTCCATACACCAAAGTTCAATCTGGTTCATTGAACA ATGGTGATGTTGCATGGTTTTTGAACGGTAAATTGAATGCATCATACAATTGTGTTGACA GACATGCCTTTGCTAATCCCGACAAGCCAGCTTTGATCTATGAAGCTGATGACGAATCCG ACAACAAAATCATCACATTTGGTGAATTACTCAGAAAAGTTTCCCAAATCGCTGGTGTCTT AAAAAGCTGGGGCGTTAAGAAAGGTGACACAGTGGCTATCTATTTGCCAATGATTCCAG AAGCGGTCATTGCTATGTTGGCTGTGGCTCGTATTGGTGCTATTCACTCTGTTGTCTTTGC TGGGTTCTCCGCTGGTTCGTTGAAAGATCGTGTCGTTGACGCTAATTCTAAAGTGGTCA TCACTTGTGATGAAGGTAAAAGAGGTGGTAAGACCATCAACACTAAAAAAATTGTTGACG AAGGTTTGAACGGAGTCGATTTGGTTTCCCGTATCTTGGTTTTCCAAAGAACTGGTACTG AAGGTATTCCAATGAAGGCCGGTAGAGATTACTGGTGGCATGAGGAGGCCGCTAAGCAG AGAACTTACCTACCTCCTGTTTCATGTGACGCTGAAGATCCTCTATTTTTATTATACACTTC CGGTTCCACTGGTTCTCCAAAGGGTGTCGTTCACACTACAGGTGGTTATTTATTAGGTG CCGCTTTAACAACTAGATACGTTTTTGATATTCACCCAGAAGATGTTCTCTTCACTGCCGG TGACGTCGGCTGGATCACGGGTCACACCTATGCTCTATATGGTCCATTAACCTTGGGTA CCGCCTCAATAATTTTCGAATCCACTCCTGCCTACCCAGATTATGGTAGATATTGGAGAAT TATCCAACGTCACAAGGCTACCCATTTCTATGTGGCTCCAACTGCTTTAAGATTAATCAA ACGTGTAGGTGAAGCCGAAATTGCCAAATATGACACTTCCTCATTACGTGTCTTGGGTT CCGTCGGTGAACCAATCTCTCCAGACTTATGGGAATGGTATCATGAAAAAGTGGGTAACA AAAACTGTGTCATTTGTGACACTATGTGGCAAACAGAGTCTGGTTCTCATTTAATTGCTCC TTTGGCAGGTGCTGTCCCAACAAAACCTGGTTCTGCTACCGTGCCATTCTTTGGTATTA ACGCTTGTATCATTGACCCTGTTACAGGTGTGGAATTAGAAGGTAATGATGTCGAAGGTG TCCTTGCCGTTAAATCACCATGGCCATCAATGGCTAGATCTGTTTGGAACCACCACGACC GTTACATGGATACTTACTTGAAACCTTATCCTGGTCACTATTTCACAGGTGATGGTGCTG GTAGAGATCATGATGGTTACTACTGGATCAGGGGTAGAGTTGACGACGTTGTAAATGTTT CCGGTCATAGATTATCCACATCAGAAATTGAAGCATCTATCTCAAATCACGAAAACGTCTC GGAAGCTGCTGTTGTCGGTATTCCAGATGAATTGACCGCTCAAACCGTCGTTGCATATG TTTCCCTAAAAGATGGTTATCTACAAAACAACGCTACTGAAGGTGATGCAGAACACATCA CACCAGATAATTTACGTAGAGAATTGATCTTACAAGTTAGGGGTGAGATTGGTCCTTTCG CCTCACCAAAAACCATTATTCTAGTTAGAGATCTACCAAGAACAAGGTCAGGAAAGATTA TGAGAAGAGTTCTAAGAAAGGTTGCTTCTAACGAAGCCGAACAGCTAGGTGACCTAACTA CTTTGGCCAACCCAGAAGTTGTACCTGCCATCATTTCTGCTGTAGAGAACCAATTTTTCTC TCAAAAAAAGAAATAA S. cerevisiae ADH1 SEQ ID NO: 19 ATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCACGGTAAGT TGGAATACAAAGATATTCCAGTTCCAAAGCCAAAGGCCAACGAATTGTTGATCAACGTTA AATACTCTGGTGTCTGTCACACTGACTTGCACGCTTGGCACGGTGACTGGCCATTGCCAG TTAAGCTACCATTAGTCGGTGGTCACGAAGGTGCCGGTGTCGTTGTCGGCATGGGTGAA AACGTTAAGGGCTGGAAGATCGGTGACTACGCCGGTATCAAATGGTTGAACGGTTCTTGTA TGGCCTGTGAATACTGTGAATTGGGTAACGAATCCAACTGTCCTCACGCTGACTTGTCTG GTTACACCCACGACGGTTCTTTCCAACAATACCCTACCCCTGACGCTGTTCAAGCCGCTC ACATTCCTCAAGGTACCGACTTGGCCCAAGTCGCCCCCATCTTGTGTGCTGGTATCACCG TCTACAAGGCTTTGAAGTCTGCTAACTTGATGGCCGGTCACTGGGTTGCTATCTCCGGTG CTGCTGGTGGTCTAGGTTCTTTGGCTGTTCAATACGCCAAGGCTATGGGTTACAGAGTCT TGGGTATTGACGGTGGTGAAGGTAAGGAAGAATTATTCAGATCCATCGGTGGTGAAGTCT TCATTGACTTCACTAAGGAAAAGGACATTGTCGGTGCTGTTCTAAAGGCCACTGACGGTG GTGCTCACGGTGTCATCAACGTTTCCGTTTCCGAAGCCGCTATTGAAGCTTCTACCAGAT ACGTTAGAGCTAACGGTACCACCGTTTTGGTCGGTATGCCAGCTGGTGCCAAGTGTTGTT CTGATGTCTTCAACCAAGTCGTCAAGTCCATCTCTATTGTTGGTTCTTACGTCGGTAACAG AGCTGACACCAGAGAAGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCA AGGTTGTCGGCTTGTCTACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCG TTGGTAGATACGTTGTTGACACTTCTAAATA S. cerevisiae ADH2 SEQ ID NO: 20ATGTCTATTCCAGAAACTCAAAAAGCCATTATCTTCTACGAATCCAACGGCAAGTT GGAGCATAAGGATATCCCAGTTCCAAAGCCAAAGCCCAACGAATTGTTAATCAACGTCA AGTACTCTGGTGTCTGCCACACCGATTTGCACGCTTGGCATGGTGACTGGCCATTGCCAA CTAAGTTACCATTAGTTGGTGGTCACGAAGGTGCCGGTGTCGTTGTCGGCATGGGTGAAA ACGTTAAGGGCTGGAAGATCGGTGACTACGCCGGTATCAAATGGTTGAACGGTTCTTGTA TGGCCTGTGAATACTGTGAATTGGGTAACGAATCCAACTGTCCTCACGCTGACTTGTCTG GTTACACCCACGACGGTTCTTTCCAAGAATACGCTACCGCTGACGCTGTTCAAGCCGCTC ACATTCCTCAAGGTACTGACTTGGCTGAAGTCGCGCCAATCTTGTGTGCTGGTATCACCG TATACAAGGCTTTGAAGTCTGCCAACTTGAGAGCAGGCCACTGGGCGGCCATTTCTGGTG CTGCTGGTGGTCTAGGTTCTTTGGCTGTTCAATATGCTAAGGCGATGGGTTACAGAGTCT TAGGTATTGATGGTGGTCCAGGAAAGGAAGAATTGTTTACCTCGCTCGGTGGTGAAGTAT ACACTGACTTCACCAAAGAGAAGGACATTGTTAGCGCAGTCGTTAAGGCTACCAACGGCG GTGCCCACGGTATCATCAATGTTTCCGTTTCCGAAGCCGCTATCGAAGCTTCTACCAGAT ACTGTTAGGGCGAACGGTACTGTTGTCTTGGTTGGTTTGCCAGCCGGTGCAAAGTGCTCCT CTGATGTCTTCAACCACGTTGTCAAGTCTATCTCCATTGTCGGCTCTTACGTGGGGAACA GAGCTGATACCAGAGAAGCCTTAGATTTCTTTGCCAGAGGTCTAGTCAAGTCTCCAATAA AGGTAGTTGGCTTATCCAGTTTACCAGAAATTTACGAAAAGATGGAGAAGGGCCAAATTG CTGGTAGATACGTTGTTGACACTTCTAAATAA S. cerevisiae ADH3 SEQ ID NO: 21 ATGTTGAGAACGTCAACATTGTTCACCAGGCGTGTCCAACCAAGCCTATTTTCTA GAAACATTCTTAGATTGCAATCCACAGCTGCAATCCCTAAGACTCAAAAAGGTGTCATCTT TTATGAGAATAAGGGGAAGCTGCATTACAAAGATATCCCTGTCCCCGAGCCTAAGCCAA ATGAAATTTTAATCAACGTTAAATATTCTGGTGTATGTCACACCGATTTACATGCTTGGCA CGGCGATTGGCCATTACCTGTTAAACTACCATTAGTAGGTGGTCATGAAGGTGCTGGTG TAGTTGTCAAACTAGGTTCCAATGTCAAGGGCTGGAAAGTCGGTGATTTAGCAGGTATCA AATGGCTGAACGGTTCTTGTATGACATGCGAATTCTGTGAATCAGGTCATGAATCAAATT GTCCAGATGCTGATTTATCTGGTTACACTCATGATGGTTCTTTCCAACAATTTGCGACCGC TGATGCTATTCAAGCCGCCAAAATTCAACAGGGTACCGACTTGGCCGAAGTAGCCCCAA TATTATGTGCTGGTGTTACTGTATATAAAGCACTAAAAGAGGCAGACTTGAAAGCTGGTG ACTGGGTTGCCATCTCTGGTGCTGCAGGTGGCTTGGGTTCCTTGGCCGTTCAATATGCAA CTGCGATGGGTTACAGAGTTCTAGGTATTGATGCAGGTGAGGAAAAGGAAAAACTTTTCA AGAAATTGGGGGGTGAAGTATTCATCGACTTTACTAAAACAAAGAATATGGTTTCTGACA TTCAAGAAGCTACCAAAGGTGGCCCTCATGGTGTCATTAACGTTTCCGTTTCTGAAGCCG CTATTTCTCTATCTACGGAATATGTTAGACCATGTGGTACCGTCGTTTTGGTTGGTTTGCC CGCTAACGCCTACGTTAAATCAGAGGTATTCTCTCATGTGGTGAAGTCCATCAATATCA AGGGTTCTTATGTTGGTAACAGAGCTGATACGAGAGAAGCCTTAGACTTCTTTAGCAGAG GTTTGATCAAATCACCAATCAAAATTGTTGGATTATCTGAATTACCAAAGGTTTATGACTT GATGGAAAAGGGCAAGATTTTGGGTAGATACGTCGTCGATACTAGTAAATAA S. cerevisiae ADH4SEQ ID NO: 22 ATGTCTTCCGTTACTGGGTTTTACATTCCACCAATCTCTTTCTTTGGTGAAGGTGCTTTA GAAGAAACCGCTGATTACATCAAAAACAAGGATTACAAAAAGGCTTTGATCGTTA CTGATCCTGGTATTGCAGCTATTGGTCTCTCCGGTAGAGTCCAAAAGATGTTGGAAGAAC GTGACTTAAACGTTGCTATCTATGACAAAACTCAACCAAACCCAAATATTGCCAATGTCAC AGCTGGTTTGAAGGTTTTGAAGGAACAAAACTCTGAAATTGTTGTTTCCATTGGTGGTG GTTCTGCTCACGACAATGCTAAGGCCATTGCTTTATTGGCTACTAACGGTGGGGAAATCG GAGACTATGAAGGTGTCAATCAATCTAAGAAGGCTGCTTTACCACTATTTGCCATCAACA CTACTGCTGGTACTGCTTCCGAAATGACCAGATTCACTATTATCTCTAATGAAGAAAAGA AAATCAAGATGGCTATCATTGACAACAACGTCACTCCAGCTGTTGCTGTCAACGATCCAT CTAACATGTTTGGTTTGCCACCTGCTTTGACTGCTGCTACTGGTCTAGATGCTTTGACTCA CTGTATCGAAGCTTATGTTTCCACCGCCTCTAACCCAATCACCGATGCCTGTGCTTTGA AGGGTATTGATTTGATCAATGAAAGCTTAGTCGCGCATACAAAGACGGTAAAGACAAGA AGGCCAGAACTGACATGTGTTACGCTGAATACTTGGCAGGTATGGCTTTCAACAANGCTT CTCTAGGTTATGTTCATGCCCTTGCTCATCAACTTGGTGGTTTCTACCACTTGCCTCATGG TGTTTGTAACGCTGTCTTGTTGCCTCATGTTCAAGAGGCCAACATGCAATGTCCAAAGG CCAAGAAGAGATTAGGTGAAATTGCTTTGCATTTCGGTGCTTCTCAAGAAGATCCAGAAG AAACCATCAAGGCTTTGCACGTTTTAAACAGAACCATGAACATTCCAAGAAACTTGAAAG AATTAGGTGTTAAAACCGAAGATTTTGAAATTTTGGCTGAACACGCCATGCATGATGCCT GCCATTTGACTAACCCAGTTCAATTCACCAAAGAACAAGTGGTTGCCATTATCAAGAAAG CCTAT GAATATTAAS. cerevisiae ADH5 SEQ ID NO: 23 ATGCCTTCGCAAGTCATTCCTGAAAAACAAAAGGCTATTCSTCTTTTATGAGACAG ATGGAAAATTGGAATATAAAGACGTCACAGTTCCGGAACCTAAGCCTAACGAAATTTTAG TCCACGTTAAATATTCTGGTGTTTGTCATAGTGACTTGCACGCGTGGCACGGTGATTGGC CATTTCAATTGAAATTTCCATTAATCGGTGGTCACGAAGGTGCTGGTGTTGTTGTTAAGT TGGGATCTAACGTTAAGGGCTGGAAAGTCGGTGATTTTGCAGGTATAAAATGGTTGAATG GGACTTGCATGTCCTGTAATATTGTGAAGTAGGTAATGAATCTCAATGTCCTTATTTGGA TGGTACTGGCTTCACACATGATGGTACTTTTCAAGAATACGCAACTGCCGATGCCGTTC AAGCTGCCCATATTCCACCAAACGTCAATCTTGCTGAAGTTGCCCCAATCTTGTGTTGCAG GTATCACTGTTTATAAGGCGTTGAAAAGAGCCAATGTGATACCAGGCCAATGGGTCACTA TATCCGGTGCATGCGGTGGCTTGGGTTCTCTGGCAATCCAATACGCCCTTGCTATGGGTT ACAGGGTCATTGGTATCGATGGTGGTAATGCCAAGCGAAAGTTATTTGAACAATTAGGCG GAGAAATATTCATCGATTTCACGGAAGAAAAAGACATTGTTGGTGCTATAATAAAGGCCA CTAATGGCGGTTCTCATGGAGTTATTAATGTGTCTGTTTCTGAAGCAGTATCGAGGCTT CTACGAGGTATTGTAGGCCCAATGGTACTGTCGTCCTGGTTGGTATGCCAGCTCATGCTT ACTGCAATTCCGATGTTTTCAATCAAGTTGTAAAATCAATCTCCATCGTTGGATCTTGTGT TGGAAATAGAGCTGATACAAGGGAGGCTTTAGATTTCTTCGCCAGAGGTTTGATCAAAT CTCCGATCCACTTAGCTGGCCTATCGGATGTTCCTGAAATTTTTGCAAAGATGGAGAAGG GTGAAATTGTTGGTAGATATGTTGTTGAGACTTCTAAATGA S. cerevisiae GPD1 SEQ ID No 24:ATGTCTGCTGCTGCTGATAGATTAAACTTAACTTCCGGCCACTTGAATGCTGGTA GAAAGAGAAGTTCCTCTTCTGTTTCTTTGAAGGCTGCCGAAAAGCCTTTCAAGGTTACTGT GATTGGATCTGGTAACTGGGTACTACTATTGCCAAGGTGGTTGCCGAAAATTGTAAGG GATACCCAGAAGTTTTCGCTCCAATAGTACAAATGTGGGTGTTCGAAGAAGAGATCAATG GTGAAAAATTGACTGAAATCATAAATACTAGACATCAAAACGTGAAATACTTGCCTGGCA TCACTCTACCCGACAATTTGGTTGCTAATCCAGACTTGATTGATTCAGTCAAGGATGTCG ACATCATCGTTTTCAACATTCCACATCAATTTTTGCCCCGTATCTGTAGCCAATTGAAAGG TCATGTTGATTCACACGTCAGAGCTATCTCCTGTCTAAAGGGTTTTGAAGTTGGTGCTA AAGGTGTCCAATTGCTATCCTCTTACATCACTGAGGAACTAGGTATTCAATGTGGTGCTCT ATCTGGTGCTAACATTGCCACCGAAGTCGCTCAAGAACACTGGTCTGAAACAACAGTTG CTTACCACATTCCAAAGGATTTCAGAGGCGAGGGCAAGGACGTCGACCATAAGGTTCTAA AGGCCTTGTTCCACAGACCTTACTTCCACGTTAGTGTCATCGAAGATGTTGCTGGTATCTC CATCTGTGGTGCTTTGAAGAACGTTGTTGCCTTAGGTTGTGGTTTCGTCGAAGGTCTAG GCTGGGGTAACAACGCTTCTGCTGCCATCCAAAGAGTCGGTTTGGGTGAGATCATCAGAT TCGGTCAAATGTTTTTCCCAGAATCTAGAGAAGAAACATACTACCAAGAGTCTGCTGGTG TTGCTGATTTGATCACCACCTGCGCTGGTGGTAGAAACGTCAAGGTTGCTAGGCTAATGG CTACTTCTGGTAAGGACGCCTGGGAATGTGAAAAGGAGTTGTTGAATGGCCAATCCGCTC AAGGTTTAATTACCTGCAAAGAAGTTCACGAATGGTTGGAAACATGTGGCTCTGTCGAAG ACTTCCCATTATTTGAAGCCGTATACCAAATCGTTTACAACAACTACCCAATGAAGAACCT GCCGGACATGATTGAAGAATTAGATCTACATGAAGATTAG S. cerevisiae GPD2 SEQ ID NO: 25: ATGCTTGCTGTCAGAAGATTAACAAGATACACATTCCTTAAGCGAACGCATCCGG TGTTATATACTCGTCGTGCATATAAAATTTTGCCTTCAAGATCTACTTTCCTAAGAAGATC ATTATTACAAACACAACTGCACTCAAAGATGACTGCTCATACTAATATCAAACAGCACA AACACTGTCATGAGGACCATCCTATCAGAAGATCGGACTCTGCCGTGTCAATTGTACATT TGAAACGTGCGCCCTTCAAGGTTACAGTGATTGGTTCTGGTAACTGGGGGACCACCATCG CCAAAGTCATTGCGGAAAACACAGAATTGCATTCCCATATCTTCGAGCCAGAGGTGAGAA TGTGGGTTTTTGATGAAAAGATCGGCGACGAAAATCTGACGGATATCATAAATACAAGAC ACCAGAACGTTAAATATCTACCCAATATTGACCTGCCCCATAATCTAGTGGCCGATCCTGA TCTTTTACACTCCATCAAGGGTGCTGACATCCTTGTTTTCAACATCCCTCATCAATTTTT ACCAAACATAGTCAAACAATTGCAAGGCCACGTGGCCCCTCATGTAAGGGCCATCTCGT GTCTAAAAGGGTTCGAGTTGGGCTCCAAGGGTGTGCAATTGCTATCCTCCTATGTTACTG ATGAGTTAGGAATCCAATGTGGCGCACTATCTGGTGCAAACTTGGCACCGGAAGTGGCCA AGGAGCATTGGTCCGAAACCACCGTGGCTTACCAACTACCAAAGGATTATGAAGGTGATG GCAAGGATGTAGATCATAAGATTTTGAAATTGCTGTTCCACAGACCTTACTTCCACGTCAA TGTCATCGATGATGTTGCTGGTATATCCATTGCCGGTGCCTTGAAGAACGTCGTGGCAC TTGCATGTGGTTTCGTAGAAGGTATGGGATGGGGTAACAATGCCTCCGCAGCCATTCAAA GGCTGGGTTTAGGTGAAATTATCAAGTTCGGTAGAATGTTTTTCCCAGAATCCAAAGTCG AGACCTACTATCAAGAATCCGCTGGTGTTGCAGATCTGATCACCACCTGCTCAGGCGGTA GAAACGTCAAGGTTGCCACATACATGGCCAAGACCGGTAAGTCAGCCTTGGAAGCAGAAA AGGAATTGCTTAACGGTCAATCCGCCCAAGGGATAATCACATGCAGAGAAGTTCACGAGT GGCTACAAACATGTGAGTTGACCCAAGAATTCCCATTATTCGAGGCAGTCTACCAGATAG TCTACAACAACGTCCGCATGGAAGACCTACCGGAGATGATTGAAGAGCTAGACATCGATG ACGAA TAGS. cerevisiae GPP1 SEQ ID NO 26:ATGCCTTTGACCACAAAACCTTTATCTTTGAAAATCAACGCCGCTCTATTCGATGT TGACGGTACCATCATCATCTCTCAACCAGCCATTGCTGCTTTCTGGAGAGATTTCGGTA AAGACAAGCCTTACTTCGATGCCGAACACGTTATTCACATCTCTCACGGTTGGAGAACTT ACGATGCCATTGCCAAGTTCGCTCCAGACTTTGCTGATGAAGAATACGTTAACAAGCTAG AAGGTGAAATCCCAGAAAAGTACGGTGAACACTCCATCGAAGTTCCAGGTGCTGTCAAGT TGTGTAATGCTTTGAACGCCTTGCCAAAGGAAAAATGGGCTGTCGCCACCTCTGGTACCC GTGACATGGCCAAGAAATGGTTCGACATTTTGAAGATCAAGAGACCAGAATACTTCATCA CCGCCAATGATGTCAAGCAAGGTAAGCCTCACCCAGAACCATACTTAAAGGGTAGAAACG GTTTGGGTTTCCCAATTAATGAACAAGACCCATCCAAATCTAAGGTTGTTGTCTTTGAAG ACGCACCAGCTGGTATTGCTGCTGGTAAGGCTGCTGGCTGTAAAATCGTTGGTATTGCTA CCACTTTCGATTTGGACTTCTTGAAGGAAAAGGGTTGTGACATCATTGTCAAGAACCACG AATCTATCAGAGTCGGTGAATACAACGCTGAAACCGATGAAGTCGAATTGATCTTTGATG ACTACTTATACGCTAAGGATGACTTGTTGAAATGGTAA S. cerevisiae GPP2 SEQ ID NO: 27 ATGGGATTGACTACTAAACCTCTATCTTTGAAAGTTAACGCCGCTTTGTTCGACGT CGACGGTACCATTATCATCTCTCAACCAGCCATTGCTGCATTCTGGAGGGATTTCGGTA AGGACAAACCTTATTTCGATGCTGAACACGTTATCCAAGTCTCGCATGGTTGGAGAACGT TTGATGCCATTGCTAAGTTCGCTCCAGACTTTGCCAATGAAGAGTATGTTAACAAATTAG AAGCTGAAATTCCGGTCAAGTACGGTGAAAAATCCATTGAAGTCCCAGGTGCAGTTAAGC TGTGCAACGCTTTGAACGCTCTACCAAAAGAGAAATGGGCTGTGGCAACTTCCGGTACCC GTGATATGGCACAAAAATGGTTCGAGCATCTGGGAATCAGGAGACCAAAGTACTTCATTA CCGCTAATGATGTCAAACAGGGTAAGCCTCATCCAGAACCATATCTGAAGGGCAGGAATG GCTTAGGATATCCGATCAATGAGCAAGACCCTTCCAAATCTAAGGTAGTAGTATTTGAAG ACGCTCCAGCAGGTATTGCCGCCGGAAAAGCCGCCGGTTGTAAGATCATTGGTATTGCCA CTACTTTCGACTTGGACTTCCTAAAGGAAAAAGGCTGTGACATCATTGTCAAAAACCACG AATCCATCAGAGTTGGCGGTACAATGCCGAAACAGACGAAGTTGAATTCATTTTTGACG ACTACTTATATGCTAAGGACGATCTGTTGAAATGGTAAdmpF ORF synthetic codon optimized for Saccharomyces cerevisiae.Reference sequence: Pseudomonas sp. CF600EMBL Accession number: X60835.1 SEQ ID NO: 28ATGAATCAGAAACTGAAAGTAGCTATCATAGGTTCCGGTAATATCGGAACAGACCTGATGATTAAGGTACTGCGTAATGCAAAGTACTTAGAAATGGGCGCGATGGTCGGTATCGATGCAGCCTCTGATGGACTGGCCAGAGCTCAAAGAATGGGCGTTACGACTACTTATGCAGGTGTAGAAGGGCTAATCAAGCTTCCTGAATTTGCAGACATAGATTTCGTCTTCGATGCTACATCTGCATCAGCCCACGTTCAGAACGAGGCTTTATTAAGACAAGCTAAACCTGGTATTAGATTGATCGACCTTACCCCGGCGGCAATCGGTCCTTACTGTGTCCCCGTAGTTAATCTCGAGGAACATTTGGGCAAGTTGAACGTTAACATGGTTACTTGCGGTGGCCAAGCTACTATTCCGATGGTCGCAGCTGTCTCACGTGTAGCCAAAGTCCATTATGCTGAGATTGTTGCTTCTATTTCAAGCAAGAGTGCCGGACCTGGAACCAGAGCCAATATAGATGAATTCACTGAGACAACCAGTAAAGCCATAGAAGTTATTGGTGGTGCTGCAAAGGGTAAAGCTATAATTATTATGAACCCAGCTGAACCACCATTGATTATGAGGGATACGGTGTATGTGCTTTCCGCCGCCGCTGATCAAGCCGCTGTCGCAGCTTCTGTGGCTGAAATGGTTCAAGCGGTTCAAGCATACGTGCCAGGCTATAGGTTAAAACAACAGGTTCAGTTTGACGTGATTCCCGAGTCCGCGCCACTAAACATCCCCGGTTTGGGGAGATTCAGCGGGTTGAAAACAAGTGTGTTCCTAGAAGTAGAAGGTGCTGCTCATTATTTGCCAGCATACGCAGGAAACTTAGATATTATGACTTCCGCAGCGTTAGCTACAGCCGAACGTATGGCGCAATCAATGTTGAATGCATAG dmpF ORF SEQ ID NO: 29MNQKLKVAIIGSGNIGTDLMIKVLRNAKYLEMGAMVGIDAASDGLARAQRMGVTTTYAGVEGLIKLPEFADIDFVFDATSASAHVQNEALLRQAKPGIRLIDLTPAAIGPYCVPVVNLEEHLGKLNVNMVTCGGQATIPMVAAVSRVAKVIIYAEIVASISSKSAGPGTRANIDEFTETTSKAIEVIGGAAKGKAIIIMNPAEPPLIMRDTVYVLSAAADQAAVAASVAEMVQAVQAYVPGYRLKQQVQFDVIPESAPLNIPGLGRFSGLKTSVFLEVEGAAHYLPAYAGNLDIMTSAALA TAERMAQSMLNAprimer dmpF-FW SEQ ID NO: 30 CATTGATTGCGCCATACG primer dmpF-RVSEQ ID NO: 31 CCGGTAATATCGGAACAGACWild type sequence of dmpF gene from Pseudomonas sp. CF600.EMBL Accession number: X60835.1 SEQ ID NO: 32 ATGAACCAGAAACTCAAAGTCGCGATCATCGGTTCGGGCAATATCGGCACCGACCTGATGATCAAGGTGCTGCGCAACGCCAAGTACCTGGAAATGGGCGCCATGGTCGGCATCGACGCCGCCTCCGACGGCCTGGCCCGCGCCCAGCGCATGGGCGTGACGACCACCTATGCCGGCGTCGAAGGGCTGATCAAGCTGCCCGAATTCGCCGACATCGATTTCGTCTTCGACGCCACCTCGGCCAGTGCCCACGTGCAGAACGAGGCGCTGCTGCGCCAGGCCAAACCTGGCATCCGCCTGATCGACCTGACCCCGGCGGCCATCGGCCCGTACTGCGTGCCGGTGGTCAATCTGGAGGAGCACCTCGGCAAGCTCAACGTCAACATGGTTACCTGCGGCGGCCAGGCGACCATCCCGATGGTCGCCGCGGTCTCCCGTGTGGCCAAGGTCCATTACGCCGAGATCGTCGCCTCGATCAGCAGCAAGTCGGCCGGACCCGGCACCCGCGCCAACATCGACGAGTTCACCGAGACCACCAGCAAAGCCATCGAAGTGATCGGTGGTGCGGCCAAGGGCAAGGCGATCATCATCATGAACCCGGCTGAGCCGCCGCTGATCATGCGCGACACCGTGTATGTGCTGTCCGCCGCCGCCGATCAGGCCGCCGTCGCGGCCTCGGTGGCGGAAATGGTTCAGGCGGTGCAGGCCTACGTGCCCGGCTATCGCCTGAAGCAGCAGGTGCAGTTCGACGTGATCCCCGAGTCCGCGCCGCTGAACATCCCCGGTCTCGGCCGGTTCAGCGGGTTGAAGACCTCGGTGTTCCTCGAAGTCGAAGGCGCCGCCCATTACCTGCCGGCCTACGCCGGCAACCTCGACATCATGACCTCCGCCGCGCTGGCTACCGCCGAGCGTATGGCGCAGTC GATGTTGAACGCCTGA

What is claimed is:
 1. A method to prepare ethanol from acetate and afermentable carbohydrate which comprises culturing yeast cells underanaerobic conditions, wherein: said culturing is carried out in afermentation medium comprising acetate and carbohydrate, wherein atleast part of the carbohydrate and at least part of the acetate has beenobtained from bagasse, sugar beet pulp or sugar beet molasses or fromstarch containing waste products from corn dry milling processes, fromcorn wet milling processes or from starch waste processes; and the yeastcells are transgenic yeast cells comprising: a recombinant heterologousnucleic acid sequence encoding a protein comprising NAD⁺-dependentacetylating acetaldehyde dehydrogenase activity (EC 1.2.1.10), andNAD⁺-dependent alcohol dehydrogenase activity (EC 1.1.1.1), and anucleic acid sequence encoding a protein with acetyl-Coenzyme Asynthetase activity (EC 6.2.1.1), and a nucleic acid sequence encoding aprotein with NADH-dependent glycerol-3-phosphate dehydrogenase activity(EC 1.1.1.8); and the transgenic yeast cells reduce the amount ofglycerol in the spent medium as compared to their correspondingwild-type yeast cells.
 2. The method of claim 1, wherein said culturingis carried out in a fermentation medium comprising acetate andcarbohydrate in a molar ratio of 0.7 or less.
 3. The method of claim 1,wherein the protein comprising NAD⁺-dependent acetylating acetaldehydedehydrogenase activity (EC 1.2.1.10) is an adhE enzyme.
 4. The method ofclaim 3, wherein the adhE enzyme is from E. coli, S. aureus, orPiromyces sp. E2.
 5. The method of claim 1, wherein the proteincomprising NAD⁺-dependent acetylating acetaldehyde dehydrogenaseactivity (EC 1.2.1.10) is an adh2 enzyme from Entamoeba histolytica. 6.The method of claim 1, wherein the nucleic acid sequence encoding theprotein with acetyl-Coenzyme A synthetase activity (EC 6.2.1.1) isrecombinant and heterologous.
 7. The method of claim 1, wherein therecombinant yeast cells are further comprising a genomic mutation in atleast one gene selected from the group consisting of GPD1 and GPD2. 8.The method of claim 7, wherein the genomic mutation comprises a mutationin the gene sequence that encodes a gpd1 enzyme.
 9. The method of claim7, wherein the genomic mutation comprises a mutation in the genesequence that encodes a gpd2 enzyme.
 10. The method of claim 7, whereinthe genomic mutation comprises a mutation in the GPD1 promoter sequence.11. The method of claim 7, wherein the genomic mutation comprises amutation in the GPD2 promoter sequence.
 12. The method of claim 1,wherein the spent medium comprises a molar ratio of glycerol to ethanolof 0.001:1 or more.
 13. The method of claim 1, wherein the spent mediumcomprises a molar ratio of glycerol:ethanol of less than 0.04:1.
 14. Themethod of claim 1, wherein, compared to its corresponding wild-typeyeast cells, the transgenic yeast cells exhibit increased acetate and/oracetic acid consumption.
 15. The method of claim 1, wherein the molarratio of acetate and/or acetic acid to carbohydrate consumed is at least0.004.
 16. The method of claim 1, wherein the method to prepare ethanolis carried out in a medium comprising acetate in the range of 0.5 to 20g/L.